Ginger metabolites and uses thereof

ABSTRACT

The present application generally relates to the use of metabolites of ginger and analogs thereof for the treatment and prevention of diseases, including but not limited to, cancer.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. Nos. 61/739,169 filed Dec. 19, 2012and 61/790,291, filed Mar. 15, 2013, the disclosure of each of which isincorporated herein by reference in its entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with Government support under CA138277 awardedby the National Institutes of Health National Cancer Institute andOffice of Dietary Supplements. The United States Government has certainrights in this invention.

FIELD

The present inventions relate generally to metabolites of shogaolcomponents of ginger, including compounds related to 6-shogaol,8-shogaol and 10-shogaol. Such compounds generally are preferentiallytoxic to cancer cells with decreased killing of non-cancer cells. Thepresent inventions also relate generally to pharmaceutical ornutraceutical compositions comprising such compounds.

BACKGROUND

Ginger (Zingiber officinale Rosc.), a member of the Zingiberaceaefamily, has been cultivated for thousands of years as a spice and formedicinal purposes. It is believed that the major pharmacologicallyactive components of ginger are gingerols and shogaols. Shogaols,□□□-unsaturated ketones which are the dehydrated products of gingerols,are the predominant pungent constituents in dried ginger.

SUMMARY

The present application is generally directed to shogaol-relatedcompounds. For example, the present application discloses metabolites ofginger components, such as metabolites of [6]-shogaol, [8]-shogaol and[10]-shogaol, including but not limited to 5-cysteinyl-[6]-shogaol(herein referred to as “M2”); 5-cysteinyl-[8]-shogaol (herein referredto as “M2′”); and 5-cysteinyl-[10]-shogaol (herein referred to as“M2″”). Such derivatives are used as pharmaceuticals for the treatmentand prevention of diseases such as cancer, or can be used as a dietarysupplement, nutraceutical or adjunct therapy. Generally, the shogaolderivatives disclosed herein are used (a) to help control symptoms ofdisease, such as cancer; (b) to alleviate unwanted side effects of anunderlying disease or a therapy for such an underlying disease; (c) toprevent future disease; or (d) to contribute to treatment of a presentdisease.

In one aspect, the present application discloses a compound of FormulaI:

wherein n is 2, 4 or 6 or a pharmaceutically acceptable salt or hydratethereof.

In another aspect, the present application discloses compound of FormulaII:

or a pharmaceutically acceptable salt or hydrate thereofwherein R is

and any diastereomers thereof.

In one variation, the present application discloses a compound ofFormula III:

or a pharmaceutically acceptable salt or hydrate thereof.

A first aspect of the present invention is a composition comprising,consisting essentially of or consisting of a compound of Formula I, II,or III.

A second aspect of the present invention is a pharmaceutical compositioncomprising, consisting essentially of or consisting of a compound ofFormula I, II, or III and a pharmaceutically acceptable carrier.

A third aspect of the present invention is a nutraceutical compositioncomprising, consisting essentially of or consisting of a compound ofFormula I, II, or III and an acceptable carrier.

A fourth aspect of the present invention is a dietary supplementcomprising, consisting essentially of or consisting of a compound ofFormula I, II, or III and an acceptable carrier.

A fifth aspect of the present invention is a method of producing acompound of Formula I, II, or III.

A sixth aspect of the present invention is a method of preventing and/ortreating a disorder in a subject in need thereof, comprising, consistingessentially of or consisting of administering to said subject atherapeutically effective amount of a composition comprising, consistingessentially of or consisting of a compound of Formula I, II, or III.

A seventh aspect of the present invention is a method of preventingand/or treating a disorder in a subject in need thereof, comprising,consisting essentially of or consisting of administering to said subjecta pharmaceutical composition comprising, consisting essentially of orconsisting of a compound of Formula I, II, or III and a pharmaceuticallyacceptable carrier.

An eighth aspect of the present invention is a method of preventing,treating, and/or contributing to the treatment of a disorder in asubject in need thereof, comprising, consisting essentially of orconsisting of administering to said subject a composition comprising,consisting essentially of or consisting of a compound of Formula I, II,or III and an acceptable carrier. The composition can be a nutraceuticalcomposition or a dietary supplement.

A ninth aspect of the present invention is a kit for preventing,treating and/or contributing to the treatment of a disorder in a subjectcomprising, consisting essentially of or consisting of a compound, apharmaceutical composition, a nutraceutical composition or a dietarysupplement of the present invention and instructions for using thecompound, the pharmaceutical composition or the nutraceuticalcomposition.

These and other objects and aspects of the present inventions willbecome apparent to those skilled in the art after a reading of thefollowing description of the disclosure when considered with thedrawings.

It will be understood that the drawings are for the purpose ofdescribing embodiments of the present application and are not intendedto limit the inventions thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the structure of [6]-shogaol, [8]-shogaol, [10]-shogaol,and some metabolites of those compounds.

FIG. 2 shows the growth inhibitory effects of [6]-shogaol, M9, or M11 onHCT-116 (A) and H-1299 (B) cells; and effects of [6]-shogaol, M9, or M11on the induction of apoptosis in HCT-116 (C) and H-1299 (D) cells. MTTassay was used to measure the growth inhibitory effect and each value inA and B represents the mean±S.D. (n=6). TUNEL assay was used to measurethe induction of apoptosis and each value in C and D represents themean±S.E. (n=10). TUNEL-positive cells have been observed at 400× power.Ten fields per slide have been counted and averaged. Significantlydifferent from DMSO control according to the two-tailed Student's t test(*, p<0.05, **, p<0.001, and ***, p<0.0001).

FIG. 3 shows the growth inhibitory effects of [6]-shogaol and itsmetabolites (M1, M2, and M4-M13) against human colon cancer cellsHOT-116 and human normal colon cells CCD-18Co. Bar, standard error(n=6).

FIG. 4 shows the growth inhibitory effects of [6]-shogaol and itsmetabolites (M1, M2, and M4-M13) against human colon cancer cells H-1299and human normal lung cells IMR-90. Bar, standard error (n=6).

FIG. 5 shows the growth inhibitory effects of [6]-shogaol, M13, M13-1,M13-2, and a physical mixture of M13-1 and M13-2 (molar/molar=1:2)against human colon cancer cells HCT-116. Bar, standard error (n=6).

FIG. 6 shows the effects of [6]-shogaol and its metabolites M2, M6, andM13 on the induction of apoptosis in HCT-116 (A) and H-1299 (B) cellsafter 24 hours of incubation; and effects of [6]-shogaol, M2, M13, M13-1and M13-2 on the induction of apoptosis in HCT-116 after 6 hours ofincubation (C). TUNEL positive cells have been observed at 400× power.10 fields per slide have been counted and averaged. Bar, standard error;o, not significant; +, p<0.05; *, p<0.01; **: P<0.0001. All statisticaltests are unpaired Student t-test, 2 tailed, compared to DMSO or thecorresponding [6]-shogaol concentration.

FIG. 7 shows Michaelis-Menten plot of [6]-shogaol metabolism to M6 inhuman liver microsomes. [6]-Shogaol was incubated with HLM at 37° C. for30 minutes with an NADPH-regenerating system. An Eadie-Hofstee plot isshown as an insert to illustrate monophase kinetics. Data pointsrepresent the mean of triplicate determinations.

FIG. 8 shows growth inhibition and induction of apoptosis by M14, M15and [6]-shogaol against human cancer cells. Human colon cancer (HCT-116)(A) and human lung cancer (H-1299) (B) cells were incubated withrespective metabolites or [6]-shogaol for 24 hours and tested for cellviability. Values are given as percent of positive control, where n=6.Induction of apoptosis by of M14 and M15. Human colon cancer cells(HCT-116) (C) and human lung cancer cells (H-1299) (D) were incubatedwith respective metabolites or [6]-shogaol for 24 hours and tested forinduction of apoptosis. Values are given as fold induction against DMSOcontrol, where n=3.

FIG. 9 shows the change in tumor weight upon treatment of 6S, M2 andM14.

FIG. 10 shows: (A) Intracellular total GSH levels and (C) GSH/GSSG ratioin A549 treated with 10 μM of 6S for 0, 2, 4, 8 and 24 hours.[GSH]_(DMSO 0hr)=43.37±5.85 nmole/mg. (B) Intracellular total GSH levelsand (D) GSH/GSSG ratio in A549 treated with 10 μM of M2 for 0, 2, 4, 8and 24 hours. [GSH]_(DMSO 0hr)=44.46±5.16 nmole/mg. bars, SEM. *, p<0.05by Student t-test.

FIG. 11 shows (A) 6S and M2 toxicity in A549 cancer cells and IMR90normal lung cells using MTT assay, with the corresponding IC₅₀ values onthe right side table. (B) Apoptosis measured by ELISA assay in A549cells after 24 hour treatment with 10 or 20 μM of 6S. (C) Apoptosismeasured by ELISA assay in A549 cells after 24 hour treatment with 10 or20 μM of M2. bars, SEM. ‡, p<0.05; ‡‡, p<0.01 using one-way ANOVAfollowed by Bonferroni's post-test.

FIG. 12 shows (A) GSH rescue assay in A549 cells. Cells were treated for24 hours with 0, 10, 20, 40 or 80 μM of 6S or M2 in the presence orabsence of 5 mM GSH. The associated table indicates the IC₅₀ values foreach treatment. (B) Effect of pft treatment on A549 cell death aftertreatment with 20 or 40 μM of 6S for 24 hours. (C) Effect of pfttreatment on A549 cell death after treatment with 20 or 40 μM of M2 for24 hours. (D) Effect of pft treatment on A549 cell apoptosis aftertreatment with 20 μM of 6S or M2 for 24 hours. bars, SEM. * p<0.05; ***p<0.001 using a paired Student's t-test; ‡: p<0.05 using one-way ANOVAfollowed by Bonferroni's post-test.

FIG. 13 shows a xenograft experiment using A549 cells in nude mice.Animals were oral-gavaged 5×/week for 7 weeks with DMSO (control), 10 mg6S per kg body weight (6S 10), 30 mg 6S per kg body weight (6S 30), or30 mg M2 per kg body weight (M2 30). (A) Changes in tumor volume (inmm³) after 1, 2, 3, 4, 5, 6 and 7 week treatment with test compounds.(B) Wet tumor weight after 7 week treatment. * p<0.05; ** p<0.01 usingunpaired Student's t-test.

FIG. 14 shows the growth inhibitory effects of 8S, M2′, 10S and M2″against human normal colon cells CCD-18Co (A) and human colon cancercells HCT-116 (B) and HT-29 (C) treated with 8S, M2′, 10S or M2″ for 24hours at different doses (n=6) (measured by MTT assay).

FIG. 15 shows that cysteine conjugated shogaols induce apoptosis viaoxidative stress-mediated p53 pathway. Protein levels were relativelyquantitated by densitometric analysis using β-actin as a loadingcontrol. Fold induction for each marker compared to DMSO is indicatedunder the corresponding line. Induction of ROS by M2 or 6S. HCT-116 andHT-29 cells were treated with indicated concentrations of 6S or M2 fordifferent time periods, and the intracellular ROS levels were determinedas described under Materials and Methods. (* p<0.05, ** p<0.01, ***p<0.001, and **** p<0.0001)

FIG. 16 shows a dose dependent inhibition of human colon cancer cellcolony formation by M2 in HCT-116 (A) and HT-29 (B) cells. Cells weretreated with M2 (0, 1, 5, 10, 20 and 40 μM) and incubated in 6-wellplates for 2 weeks, and the cells were then stained with crystal violetand counted for colony formation. Each column represents a mean±SD(n=3; * p<0.05; ** p<0.01; and *** p<0.001)

DETAILED DESCRIPTION

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to compositions and methodologiesprovided herein.

This description is not intended to be a detailed catalogue of all theways in which the present invention may be implemented or of all thefeatures that may be added to the present invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. Thus, one ormore of the method steps included in a particular method describedherein may, in other embodiments, be omitted and/or performedindependently. In addition, numerous variations and additions to theembodiments suggested herein, which do not depart from the instantinvention, will be apparent to those skilled in the art in light of theinstant disclosure. Hence, the following description is intended toillustrate some particular embodiments of the invention, and not toexhaustively specify all permutations, combinations and variationsthereof. It should therefore be appreciated that the present inventionis not limited to the particular embodiments set forth herein. Rather,these particular embodiments are provided so that this disclosure willconvey the full scope of the invention to those skilled in the art.

The terminology used herein is for the purpose of describing particularembodiments of the present invention only and is not intended to limitthe present invention.

Although the following terms are believed to be well understood by oneof skill in the art, the following definitions are set forth tofacilitate understanding of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.

As used herein, the terms “a” or “an” or “the” may refer to one or morethan one. For example, “a” pharmaceutically acceptable excipient canmean one pharmaceutically acceptable excipient or a plurality ofpharmaceutically acceptable excipients.

As used herein, the term “about,” when used in reference to a measurablevalue such as an amount of mass, dose, time, temperature, and the like,is meant to encompass variations of +/−20% of the specified amount.

All ranges set forth, unless otherwise stated, include the statedendpoints and all increments between.

As used herein, the term “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “cancer” refers to any benign or malignantabnormal growth of cells. Examples include, without limitation, breastcancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, coloncancer, colorectal cancer, melanoma, malignant melanoma, ovarian cancer,brain cancer, primary brain carcinoma, head-neck cancer, glioma,glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer,head or neck carcinoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma,testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomachcarcinoma, colon carcinoma, prostatic carcinoma, genitourinarycarcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiplemyeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,adrenal cortex carcinoma, malignant pancreatic insulinoma, malignantcarcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,chronic myelogenous leukemia, chronic granulocytic leukemia, acutegranulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma. In some embodiments, the cancer is selected from thegroup of tumor-forming cancers. In some embodiments, the cancer iscolorectal cancer or lung cancer.

In some embodiments, the cancer is a cancer formed at a different siteof a body as a result of migration of a cell from a cancer (i.e.metastasis) including but not limited to any cancer mentioned herein.

In other embodiments, a compound or composition of the presentapplication is used for the prevention of one cancer or metastasis ofone cancer and concurrently for the treatment of another cancermentioned hereinabove.

The present application also involves the delivery of therapeuticcompounds to subjects exhibiting pre-cancerous symptoms to prevent theonset of cancer. Cells of this category include but are not limited topolyps and other precancerous lesions, premalignancies, preneoplastic orother aberrant phenotype indicating probable progression to a cancerousstate.

As used herein, the phrase “nutraceutical composition” or variantsthereof refers to compositions containing a compound disclosed hereinand further containing a food or a liquid, part of a food or a liquid,or is an addition to a food or a liquid, wherein such compositionprovides medical or health benefits, including the prevention andtreatment of disease either alone or in combination with a primarytherapy, or the trigger of a beneficial physiological response.

A nutraceutical composition as disclosed herein provides a nutritionalsource, thus, a nutraceutical composition can be a food product,foodstuff, functional food, or a supplement composition for a foodproduct or a foodstuff. As used herein, the term food product refers toany food which provides a nutritional source and is suitable for oralconsumption by humans or animals. The food product may be a prepared andpackaged food or an animal feed. As used herein, the term foodstuffrefers to a nutritional source for human or animal consumption.Functional foods are foods consumed as part of a diet which aredemonstrated to have physiological benefits beyond basic nutritionalfunctions. Food products, foodstuffs, or functional foods include butare not limited to beverages, such as non-alcoholic and alcoholic drinksas well as liquid preparations to be added to drinking water and liquidfood, and solid or semi-solid foods. Non-alcoholic drinks include butare not limited to nutritional shakes, soft drinks; sport drinks; fruitjuices; and milk and other dairy drinks such as yogurt drinks andprotein shakes. Examples of solid or semi-solid food include, but arenot limited to, baked goods; puddings; dairy products; confections;snack foods; or frozen confections or novelties; prepared frozen meals;candy; liquid food such as soups; spreads; sauces; salad dressings;prepared meat products; cheese; yogurt and any other fat or oilcontaining foods; and food ingredients.

As used herein, the term “consists essentially of” (and grammaticalvariants thereof), as applied to the compositions and methods of thepresent invention, means that the compositions/methods may containadditional components so long as the additional components do notmaterially alter the composition/method. The term “materially alter,” asapplied to a composition/method of the present invention, refers to anincrease or decrease in the effectiveness of the composition/method ofat least about 20% or more. For example, a component added to acomposition of the present invention would “materially alter” thecomposition if it increases or decreases the composition's ability toinhibit tumor growth by at least 20%.

As used herein, the term “emulsion” refers to a suspension or dispersionof one liquid within a second immiscible liquid. In some embodiments,the emulsion is an oil-in-water emulsion or a water-in-oil emulsion. Insome embodiments, “emulsion” may refer to a material that is a solid orsemi-solid at room temperature and is a liquid at body temperature(about 37° C.).

As used herein, “pharmaceutically acceptable” means that the material issuitable for administration to a subject and will allow desiredtreatment to be carried out without giving rise to unduly deleteriousadverse effects. The severity of the disease and the necessity of thetreatment are generally taken into account when determining whether anyparticular side effect is unduly deleterious.

As used herein the terms “purified,” or “isolated” refer to a compoundafter isolated from a synthetic process (e.g., from a reaction mixture),or from a natural source or some combination thereof. Thus, the term“purified,” or its alternatives, including “in purified form” or “inisolated and purified form” refers to the physical state of a compoundafter being obtained from a purification process or processes asdescribed herein or well known to the skilled artisan (e.g.,chromatography, recrystallization and the like), in sufficient purity tobe characterizable by standard analytical techniques described herein orwell known to the skilled artisan. The purification techniques disclosedherein result in isolated and purified forms of the subject metabolites.Such isolation and purification techniques would be expected to resultin product purities of 95 wt % or better, including enantiomers of thesame molecule.

As used herein, the terms “prevent,” “preventing,” and “prevention” (andgrammatical variants thereof) refer to avoidance, prevention and/ordelay of the onset of a disease, disorder and/or a clinical symptom(s)in a subject and/or a reduction in the severity of the onset of thedisease, disorder and/or clinical symptom(s) relative to what wouldoccur in the absence of the compositions and/or methods of the presentinvention. In some embodiments, prevention is complete, resulting in thetotal absence of the disease, disorder and/or clinical symptom(s). Insome embodiments, prevention is partial, resulting in reduced severityand/or delayed onset of the disease, disorder and/or clinicalsymptom(s).

As used herein, the term “prevention effective amount” (and grammaticalvariants thereof) refers an amount that is sufficient to prevent and/ordelay the onset of a disease, disorder and/or clinical symptoms in asubject and/or to reduce and/or delay the severity of the onset of adisease, disorder and/or clinical symptoms in a subject relative to whatwould occur in the absence of the methods of the invention. Thoseskilled in the art will appreciate that the level of prevention need notbe complete, as long as some benefit is provided to the subject.

As used herein, the term “subject” (and grammatical variants thereof)refers to mammals, avians, reptiles, amphibians, or fish. Mammaliansubjects may include, but are not limited to, humans, non-human primates(e.g., monkeys, chimpanzees, baboons, etc.), dogs, cats, mice, hamsters,rats, horses, cows, pigs, rabbits, sheep and goats. Avian subjects mayinclude, but are not limited to, chickens, turkeys, ducks, geese, quailand pheasant, and birds kept as pets (e.g., parakeets, parrots, macaws,cockatoos, and the like). In particular embodiments, the subject is froman endangered species. In particular embodiments, the subject is alaboratory animal. Human subjects may include neonates, infants,juveniles, adults, and geriatric subjects. In particular embodiments,the subject is male. In particular embodiments, the subject is female.

As used herein, the term “therapeutically effective” refers to provisionof some improvement or benefit to the subject. Alternatively stated, a“therapeutically effective amount” is an amount that will provide somealleviation, mitigation, or decrease in at least one clinical symptom inthe subject (e.g., in the case of cancer, reduced tumor size, decreasedincidence of metastasis, etc.). Those skilled in the art will appreciatethat the therapeutic effects need not be complete or curative, as longas some benefit is provided to the subject.

As used herein, the terms “therapeutically effective amount” and“therapeutically acceptable amount” (and grammatical variants thereof)refer to an amount that will elicit a therapeutically useful response ina subject. The therapeutically useful response may provide somealleviation, mitigation, or decrease in at least one clinical symptom inthe subject. The terms also include an amount that will prevent or delayat least one clinical symptom in the subject and/or reduce and/or delaythe severity of the onset of a clinical symptom in a subject relative towhat would occur in the absence of the methods of the invention. Thoseskilled in the art will appreciate that the therapeutically usefulresponse need not be complete or curative or prevent permanently, aslong as some benefit is provided to the subject.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, inhibiting the progressof or preventing a disease or disorder. In some embodiments, treatmentmay be administered after one or more symptoms have developed. In otherembodiments, treatment may be administered in the absence of symptoms.For example, treatment may be administered to a susceptible individualprior to the onset of symptoms (e.g., in light of a history of symptomsand/or in light of genetic or other susceptibility factors). Treatmentmay also be continued after symptoms have resolved, for example, toprevent or delay their recurrence.

As used herein, the term “treatment effective amount” (and grammaticalvariants thereof) refers to an amount that is sufficient to provide someimprovement or benefit to the subject. Alternatively stated, a“treatment effective amount” is an amount that will provide somealleviation, mitigation, decrease, or stabilization in at least oneclinical symptom in the subject. Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

In one aspect of the present invention, the compositions disclosedherein are “nutraceutical compositions” help control symptoms ofdisease; alleviate unwanted side effects of an underlying disease or atherapy for such an underlying disease; prevent future disease, orcontribute to treatment of a disease. In some embodiments, thenutraceutical composition is administered at the same time as thepharmaceutical treatment; in other embodiments, nutraceuticalcomposition is administered before or after pharmaceutical treatment.When the nutraceutical composition is administered before or afterpharmaceutical treatment, such administration occurs hours, days, ormonths before pharmaceutical treatment. In some embodiments, thenutraceutical composition is administered after one or more symptomshave developed. In other embodiments, the nutraceutical composition isadministered in the absence of symptoms. For example, the nutraceuticalcomposition is administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Administration of thenutraceutical composition may also be continued after symptoms haveresolved, for example, to prevent or delay their recurrence.

The present invention provides compositions useful for the preventionand/or treatment of disease.

In one aspect, the present application discloses a compound of FormulaI:

wherein n is selected from the group of 2, 4, 6 and combinationsthereof. In one variation, the compound is a pharmaceutically acceptablesalt or hydrate thereof. In one variation, the compound is in anisolated or purified form. In one embodiment of the compound of FormulaI, n is 2; in another embodiment, n is 4 and in yet another embodiment,n is 6. As would be understood by those in the art, “2” indicates theexistence of two “n” components, “4” indicates the existence of four “n”components, and “6” indicates the existence of six “n” components.

In another aspect, the present application discloses compound of FormulaII:

or a pharmaceutically acceptable salt or hydrate thereof,

wherein R is

and any diastereomers thereof. In one variation, the compound is in anisolated or purified form. In one embodiment, the compound is:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form. In another embodiment, the compound is:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form.

In another aspect, the present application discloses a compound ofFormula III:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form.

In another aspect, the present application discloses a compound havingthe formula:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form.

In another aspect, the present application discloses a compound havingthe formula:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form.

In yet another aspect, the present application discloses a compoundhaving the formula:

or a pharmaceutically acceptable salt or hydrate thereof, in an isolatedor purified form.

Compounds of the present invention may exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. The chemical structures depicted herein are intended toencompass all possible enantiomers and stereoisomers of the illustratedcompounds, including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures. As will be understood bythose skilled in the art, enantiomeric and stereoisomeric mixtures canbe resolved into their component enantiomers or stereoisomers using wellknown separation techniques and/or chiral synthesis techniques.

Compounds of the present invention may exist in several tautomericforms, including the enol form, the keto form and mixtures thereof. Thechemical structures depicted herein are intended to encompass allpossible tautomeric forms of the illustrated compounds.

Compounds of the present invention may be stable in environments havinga pH less than about 0.1 to 14.0, or any sub-range within that range

Compounds of the present invention may exist as isotopically labeledcompounds, wherein one or more atoms have an atomic mass different fromthe atomic mass conventionally found in nature. Examples of isotopesthat can be incorporated into the compounds of the present inventioninclude, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹F,³²P, ³⁵S, ¹⁸F and ³⁶Cl. The chemical structures depicted herein areintended to encompass all possible isotopically labeled versions of thecompounds of the present invention. Isotopically labeled compounds ofthe present invention may be used in any suitable method known in theart, including, but not limited to, methods of preventing, diagnosing,monitoring and/or treating a disorder.

Compounds of the present invention may comprise any suitablepharmaceutically acceptable salt, including, but not limited to, acidaddition salts and base addition salts. Examples of suitable salts canbe found for example in Stahl and Wermuth, Handbook of PharmaceuticalSalts Properties, Selection, and Use, Wiley-VCH, Weinheim, Germany(2002); and Berge et al., “Pharmaceutical Salts,” J. of PharmaceuticalScience, 1977; 66:1-19. In some embodiments, the pharmaceuticallyacceptable salts of the compounds is a disalt. In some embodiments, thepharmaceutically acceptable salt is an L-tartrate salt.

Pharmaceutically acceptable acid addition salts include but are notlimited to non-toxic salts derived from inorganic acids such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic,phosphorus, and the like, as well as the salts derived from organicacids, such as aliphatic mono- and dicarboxylic acids,phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioicacids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Suchsalts thus include the acetate, aspartate, benzoate, besylate(benzenesulfonate), bicarbonate/carbonate, bisulfate, caprylate,camsylate (camphor sulfonate), chlorobenzoate, citrate, edisylate(1,2-ethane disulfonate), dihydrogenphosphate, dinitrobenzoate, esylate(ethane sulfonate), fumarate, gluceptate, gluconate, glucuronate,hibenzate, hydrochloride/chloride, hydrobromide/bromide,hydroiodide/iodide, isobutyrate, monohydrogen phosphate, isethionate,D-lactate, L-lactate, malate, maleate, malonate, mandelate, mesylate(methanesulfonate), metaphosphate, methylbenzoate, methylsulfate,2-napsylate (2-naphthalene sulfonate), nicotinate, nitrate, orotate,oxalate, palmoate, phenylacetate, phosphate, phthalate, propionate,pyrophosphate, pyrosulfate, saccharate, sebacate, stearate, suberate,succinate sulfate, sulfite, D-tartrate, L-tartrate, tosylate (toluenesulfonate), and xinafoate salts, and the like of compounds describedherein. Also contemplated are the salts of amino acids such as arginate,gluconate, galacturonate, and the like.

Acid addition salts of the compounds disclosed herein may be prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentapplication.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metal hydroxides, orof organic amines. Examples of metals used as cations are aluminum,calcium, magnesium, potassium, sodium, and the like. Examples ofsuitable amines include arginine, choline, chloroprocaine,N,N′-dibenzylethylenediamine, diethylamine, diethanolamine, diolamine,ethylenediamine (ethane-1,2-diamine), glycine, lysine, meglumine,N-methylglucamine, olamine, procaine (benzathine), and tromethamine.

The base addition salts of the compounds disclosed herein may beprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in a conventional manner. The free acidforms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present application.

In some embodiments, the present invention provides a compositioncomprising, consisting essentially of or consisting of one or morecompounds of the present invention.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising, consisting essentially of or consisting of 1) apharmaceutically acceptable carrier and 2) a unit dose of an activeingredient wherein said active ingredient is a compound of Formula I:

wherein n is selected from the group consisting of 2, 4, 6 andcombinations thereof. In one variation, the compound is apharmaceutically acceptable salt or hydrate thereof. In anothervariation, the compound is in an isolated or purified form.

In one embodiment of the composition comprises a compound of Formula Iwherein n is 2; in another embodiment, n is 4 and in yet anotherembodiment, n is 6. In another embodiment, the composition comprises oneor more of the compounds of Formula I, such as a composition comprisinga compound where n is 2 and a compound where n is 4, or a compound wheren is 2 and a compound where n is 6 or a compound where n is 4 and acompound where n is 6. Alternately, such a composition comprises acompound where n is 2 and a compound where n is 4 and a compound where nis 6. In one variation, the compound is in an isolated or purified form.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising, consisting essentially of or consisting of 1) apharmaceutically acceptable carrier and 2) a unit dose of an activeingredient wherein said active ingredient is a compound of Formula II:

or a pharmaceutically acceptable salt or hydrate thereof wherein R is

and any diastereomers thereof. In one variation, the compound is in anisolated or purified form.

In one variation, the pharmaceutical composition comprises:

or a pharmaceutically acceptable salt or hydrate thereof. In anothervariation, the pharmaceutical composition comprises:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising, consisting essentially of or consisting of 1) apharmaceutically acceptable carrier and 2) a compound of Formula III:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form.

In some embodiments, the present invention provides a nutraceuticalcomposition comprising, consisting essentially of or consisting of 1) afood grade carrier and 2) an active ingredient wherein said activeingredient is a compound of Formula I

wherein n is 2, 4, 6, and combinations thereof. In one variation, thecompound is a pharmaceutically acceptable salt or hydrate thereof. Inone variation, the compound is in an isolated or purified form.

In one embodiment of the composition comprises a compound of Formula Iwherein n is 2; in another embodiment, n is 4 and in yet anotherembodiment, n is 6. In another embodiment, the composition comprises oneor more of the compounds of Formula I, such a composition comprises acompound where n is 2 and a compound where n is 4, or the compositioncomprises a compound where n is 2 and a compound where n is 6, or thecomposition comprises a compound where n is 4 and a compound where n is6. Alternately, such a composition comprises a compound where n is 2, acompound where n is 4 and a compound where n is 6. In another variation,the composition comprises a compound where n consists essentially of 2and 4, or 2 and 6, or 4 and 6, or n consists essentially of 2, 4 and 6.

In another aspect, the present invention provides a nutraceuticalcomposition comprising, consisting essentially of or consisting of 1) afood grade carrier and 2) a compound of Formula II:

or a pharmaceutically acceptable salt or hydrate thereof wherein R is

and any diastereomers thereof. In one variation, the compound is in anisolated or purified form.

In one variation, the nutraceutical composition comprises:

or a pharmaceutically acceptable salt or hydrate thereof.In another variation, the nutraceutical composition comprises:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form.

In another aspect, the present invention provides a nutraceuticalcomposition comprising, consisting essentially of or consisting of 1) afood grade carrier and 2) a compound of Formula III:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form.

In another aspect, the present invention provides a compositioncomprising, consisting essentially of or consisting of 1) an acceptablecarrier and 2) one or more compounds having the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form.

In another aspect, the present invention provides a compositioncomprising, consisting essentially of or consisting of 1) an acceptablecarrier and 2) one or more compounds having the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form.

In yet another aspect, the present invention provides a compositioncomprising, consisting essentially of or consisting of 1) an acceptablecarrier and 2) one or more compounds having the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In oneembodiment, the composition comprises one or more of M9, M9′ and M9″ ora pharmaceutically acceptable salt or hydrate thereof. In anotherembodiment, the composition comprises one or more of M11, M11′ and M11″or a pharmaceutically acceptable salt or hydrate thereof. In onevariation of any disclosed aspect or embodiment, the composition doesnot comprise M2, M9 or M11. In another variation, the compositioncomprises M2, M2′, M2″, M9, M9′, M9″, M11, M11′ and M11″ or apharmaceutically acceptable salt. In yet another variation, thecomposition comprises M2′, M2″, M9′, M9″, M11′ and M11″. In onevariation, the compound is in an isolated or purified form.Formulations

Pharmaceutical compositions of the present invention may comprise anysuitable pharmaceutical carrier moiety, including, but not limited to,phosphate buffered saline and isotonic saline solution. Other examplesof pharmaceutically acceptable carriers may be found, for example, inANSEL'S PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (9th Ed.,Lippincott Williams and Wilkins (2010)), PHARMACEUTICAL SCIENCES (18thEd., Mack Publishing Co. (1990) or REMINGTON: THE SCIENCE AND PRACTICEOF PHARMACY (21st Ed., Lippincott Williams & Wilkins (2005)).

Pharmaceutical compositions of the present invention may comprise anysuitable diluent or excipient, including, but not limited to, those setforth in ANSEL'S PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS(9th Ed., Lippincott Williams and Wilkins (2010)), HANDBOOK OFPHARMACEUTICAL EXCIPIENTS (6th Ed., American Pharmaceutical Association(2009)) and REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (21st Ed.,Lippincott Williams & Wilkins (2005)). In some embodiments, thecomposition comprises one or more pharmaceutically acceptable diluentsand/or one or more pharmaceutically acceptable excipients.

Pharmaceutical compositions of the present invention may comprise anysuitable auxiliary substance, including, but not limited to, pHadjusting and/or buffering agents, tonicity adjusting and/or bufferingagents and lipid-protective agents that protect lipids againstfree-radical and lipid-peroxidative damages (e.g., alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine).

Pharmaceutical compositions containing the active ingredient may be inany form suitable for the intended method of administration and may beprepared according to any suitable method. Compositions in the form oftablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, syrups or elixirsmay be prepared.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation. Tabletscontaining the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniquesincluding microencapsulation to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil

Aqueous suspensions of the application contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the application suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the application may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oil,such as olive oil or arachis oil, a mineral oil, such as liquidparaffin, or a mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the application may be in the form ofa sterile injectable preparation, such as a sterile injectable aqueousor oleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The compositions can be administered intravenously or by catheter-basedtechniques, or a combination thereof, with or without associateddelivery devices (i.e. pumps). For example, treatment can beadministered intravenously, in or associated with cardioplegiasolutions, via local delivery procedures including direct injection intografts or native arteries, and via perfusion-assisted techniques. Thecompositions of the present application can be infused intravenously,while other therapeutically active agents are delivered to the targetorgan selectively, or both therapies can be delivered by eitherintravenous or intravascular selective administration.

As noted above, formulations of the present application suitable fororal administration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a freeflowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropyl methylcellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

The compositions described herein can be immediate-release formulations.A variety of known methods and materials may be used to bring about theimmediate release. For instance, placement of the agent along anexterior of a tablet (e.g., coating the exterior or formulating theouter layer with the agent) and/or combined with forming a tablet bycompressing the powder using low compaction can produceimmediate-release of the agent from the composition. The composition canalso be in a controlled-release form. The compositions can also be in asustained release form.

The compositions therefore can comprise one or more carriers thatprotect the agents against rapid elimination from the body, such astime-release formulations or coatings. Such carriers includecontrolled-release formulations, including, for example,microencapsulated delivery systems. Compounds of the presentapplication, a pharmaceutically acceptable salt thereof, can be includedin the pharmaceutically acceptable carrier in amounts sufficient totreat an individual. The controlled-release form can be in an amountthat is effective to protect the agent from rapid elimination from thebody, or to provide a sustained release or dosage, such as between about1 □g/kg/min to about 500 □g/kg/min, or any sub-range within. In otherembodiments, the unit dose is from about 1 to about 500 mg/kg, or anysub-range within, of compound of the present application, such as acompound of the present application, or pharmaceutically acceptable saltthereof.

In certain embodiments the compositions are in oral dosage form andcomprise a matrix that includes a controlled-release material. Incertain embodiments, the matrix is compressible into a tablet and can beoptionally overcoated with a coating that can control the release of thecompound of the present application or pharmaceutically acceptable saltthereof, from the composition. In this embodiment, the compound orpharmaceutically acceptable salt thereof, is maintained within atherapeutic range over an extended period of time. In certain alternateembodiments, the matrix is encapsulated.

Tablets or capsules containing a composition of the present applicationcan be coated or otherwise compounded to provide a dosage form affordingthe advantage of prolonged action. For example, the tablet or capsulecan contain an inner dosage and an outer dosage component, the latterbeing in the form of an envelope over the former. The two components canbe separated by an enteric layer that serves to resist disintegration inthe stomach and permit the inner component to pass intact into theduodenum or to be delayed in release. For controlled extended release,the capsule can also have micro drilled holes.

A coating comprising an initial dose or first dose of a compound of thepresent application or pharmaceutically acceptable salt thereof, inimmediate release form, can be added to the outside of acontrolled-release tablet core comprising a second dose of a compound ofthe present application or pharmaceutically acceptable salt thereof, toproduce a final dosage form. Such a coating can be prepared by admixingthe first dosage with polyvinylpyrrolidone (PVP) 29/32 or hydroxypropylmethylcellulose (HPMC) and water/isopropyl alcohol and triethyl acetate.Such an immediate-release coating can be spray coated onto the tabletcores. The immediate-release coating can also be applied using apress-coating process with a blend consisting of 80% by weightpromethazine and 20% by weight of lactose and hydroxypropylmethylcellulose type 2910. Press-coating techniques are known in theart.

The immediate-release or controlled-release dosage forms of the presentapplication can also take the form of a multilayer tablet, such as abi-layered tablet, which comprises a first layer and a second layer. Ina further aspect of the bi-layered tablet, the first layer is animmediate release layer and/or the second layer is a controlled-releaselayer. For example, a multilayered tablet can comprise at least oneimmediate release layer comprising an amount of a compound of thepresent application or pharmaceutically acceptable salt thereof and atleast one controlled release layer which comprises an amount of acompound of the present application or a pharmaceutically acceptablesalt thereof. The controlled release layer may provide sustained releaseof a compound of the present application or pharmaceutically acceptablesalt thereof, for a period of time. Alternatively, the immediate releaselayer and the controlled released layer may provide sustained release ofa compound of the present application or pharmaceutically acceptablesalt thereof, but at different dosage amounts.

The immediate-release or controlled release dosage forms of the presentapplication can also take the form of pharmaceutical particlesmanufactured by a variety of methods, including but not limited tohigh-pressure homogenization, wet or dry ball milling, or small particleprecipitation. Other methods to make a suitable powder formulation arethe preparation of a solution of active ingredients and excipients,followed by precipitation, filtration, and pulverization, or followed byremoval of the solvent by freeze-drying, followed by pulverization ofthe powder to the desired particle size. These dosage forms can includeimmediate-release particles in combination with controlled-releaseparticles in a ratio sufficient useful for delivering the desireddosages of active agents.

In another aspect of the present application, the components arereleased from a multi-layered tablet that comprise at least a firstlayer, a second layer and a third layer. Wherein, the layers containinga therapeutically active agent can be optionally separated by one ormore layers of inert materials. In one embodiment the layers containingan agent have similar rates of release, e.g. all are immediate releaseor all are controlled-release. In an alternative embodiment the layershave different rates of release. In this aspect at least one layer is animmediate release layer and at least one layer is a controlled releaselayer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Transdermal delivery systems manufactured as an adhesive disc or patchthat slowly releases the active ingredient for percutaneous absorptionmay be used. To this end, permeation enhancers may be used to facilitatetransdermal penetration of the active agent. For example, fortransdermal administration, the compounds herein may be combined withskin penetration enhancers, such as propylene glycol, polyethyleneglycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, dimethylsulfoxide, and the like, which increase the permeability of the skin tothe compounds, and permit them to penetrate through the skin and intothe bloodstream. The compounds herein may also be combined with apolymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which may be dissolved in solvent, such asmethylene chloride, evaporated to the desired viscosity, and thenapplied to backing material to provide a patch. The compounds may beadministered transdermally to achieve a local concentration of theactive agent or to achieve systemic administration of the active agent.

Generally speaking, transdermal drug delivery systems are commonlyeither reservoir-type or matrix-type devices. Both types of devicesinclude a backing layer that forms the outer surface of the finishedtransdermal device and which is exposed to the environment during use,and a release liner or protective layer that forms the inner surface andwhich covers the adhesive means for affixing the devices to the skin ormucosa of a user. The release liner or protective layer is removed priorto application, exposing the adhesive means which is typically apressure-sensitive adhesive. The active agent is located between therelease liner and backing layer, usually solubilized or dispersed in asolvent or carrier composition. In some embodiments, the outer surfaceof the transdermal device (e.g., patch) is adapted to associate with asecond component, such as a heating compartment (e.g., electrical orchemical means for providing controlled and consistent increase intemperature).

In some embodiments, the present invention provides a kit comprising,consisting essentially of or consisting of a compound or pharmaceuticalcomposition of the present invention and instructions for using thecompound or pharmaceutical composition to prevent, monitor and/or treata disorder. In some embodiments, the subject is an animal, such as ahuman. In some embodiments, the kit further comprises at least one otheragent for use in the treatment of cancer, for reducing side effectsinduced by the compound of the application, and/or for enhancing thetherapeutic efficacy of the compound of the application.

In some embodiments, the present invention provides a kit comprising,consisting essentially of or consisting of a composition of the presentinvention, a supplemental composition and instructions for using thecomposition of the present invention and the supplemental composition toprevent, monitor and/or treat a disorder.

Kits of the present invention may comprise instructions for preventing,monitoring and/or treating any suitable disorder, including, but notlimited to, cancer. In some embodiments, the disorder is agastrointestinal cancer, such as an anal cancer, an esophageal cancer, astomach cancer, a liver cancer, a gallbladder cancer, a pancreaticcancer, a colon cancer or a rectal cancer. In some embodiments, thedisorder is lung cancer.

Kits of the present invention may comprise instructions for preventing,monitoring and/or treating a disorder in any suitable subject,including, but not limited to, human subjects.

Another aspect of the present invention is a kit for preventing,treating and/or supplementing treatment of a disorder in a subjectcomprising, consisting essentially of or consisting of a compound,pharmaceutical composition or nutraceutical composition of the presentinvention and instructions for using the compound, the pharmaceuticalcomposition or the nutraceutical composition.

In one aspect, the kit includes a compound of Formula I:

wherein n is 2, 4 or 6 or a pharmaceutically acceptable salt or hydratethereof. In one embodiment of the kit comprises a compound of Formula Iwherein n is 2; in another embodiment, n is 4 and in yet anotherembodiment, n is 6. In another embodiment, the kit comprises one or moreof the compounds of Formula I, such as a kit comprising a compound wheren is 2 and a compound where n is 4, or a compound where n is 2 and acompound where n is 6 or a compound where n is 4 and a compound where nis 6. Alternately, such a kit comprises a compound where n is 2 and acompound where n is 4 and a compound where n is 6. In one variation ofany disclosed aspect or embodiment, the compound is in an isolated orpurified form; alternately the compound is in a synthetic reactionmixture.

In another aspect, the kit includes a compound of Formula II:

or a pharmaceutically acceptable salt or hydrate thereof wherein R is

and any diastereomers thereof. In one variation, the compound is in anisolated or purified form; alternately the compound is in a syntheticreaction mixture.

In one variation, the kit includes:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form. In another variation, the kit includes:

or a pharmaceutically acceptable salt or hydrate thereof in an isolatedor purified form.

In another aspect, the kit includes a compound of Formula III:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form; alternatelythe compound is in a synthetic reaction mixture.

In one aspect, the kit includes one or more compounds having theformula:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form. In anothervariation, the compound is in a synthetic reaction mixture.

In another aspect, the kit includes M14 or M15:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form. In anothervariation, the compound is in a synthetic reaction mixture.

In yet another aspect, the kit includes one or more of:

or a pharmaceutically acceptable salt or hydrate thereof. In oneembodiment, the kit includes one or more of M9, M9′ and M9″ or apharmaceutically acceptable salt or hydrate thereof. In anotherembodiment, the kit includes one or more of M11, M11′ and M11″ or apharmaceutically acceptable salt or hydrate thereof. In one variation ofany disclosed aspect or embodiment, the kit does not include M2, M9 orM11. In another variation, the kit includes M2, M2′, M2″, M9, M9′, M9″,M11, M11′ and M11″ or a pharmaceutically acceptable salt. In yet anothervariation, the kit includes M2′, M2″, M9′, M9″, M11′ and M11″. In onevariation of any aspect or embodiment, the compound is in an isolated orpurified form. In another variation, the compound is in a syntheticreaction mixture.Pharmaceutical and Nutraceutical Administration

In some embodiments, the compound is administered parenterally. In someembodiments, the compound is administered orally.

For the purposes of this application, the compounds of the presentapplication or pharmaceutically acceptable salt thereof may beadministered by a variety of means including orally, parenterally, byinhalation spray, topically, or rectally in formulations containingpharmaceutically acceptable carriers, adjuvants and vehicles. Thecompound can also be administered as depot formulations. Pharmaceuticalcompositions containing the active ingredient may be in any formsuitable for the intended method of administration.

The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, and intraarterial injections with a variety of infusiontechniques. Intraarterial and intravenous injection as used hereinincludes administration through catheters.

It will be understood that the specific dose level for any particularpatient will depend on a variety of factors including the activity ofthe specific compound employed; the age, body weight, general health,sex and diet of the individual being treated; the time and route ofadministration; the rate of excretion; other drugs which have previouslybeen administered, as is well understood by those skilled in the art.Convenient dosing includes, but is not limited to, a once a day or twicea day administration, such as a tablet or capsule, as well asintravenous infusions. The use of time-release preparations to controlthe rate of release of the active ingredient as well as continuousinfusions may also be employed. The dose may be administered in as manydivided doses as is convenient.

Unit dosage formulations can be those containing a daily dose or unit,daily sub-dose, or an appropriate fraction thereof, of compound of thepresent application or a pharmaceutically acceptable salt thereof. Theunit dose may be for oral consumption, such as by a tablet or capsule,or for infusion, or administered by other means as disclosed herein. Insome embodiments, the dose amount is provided once a day, twice a day, 3times a day, or 4 or more times a day. In other embodiments, the doseamount is provided twice a week, once a week, twice a month or once amonth. For example, a dose can be provided twice a day, 3 times a day,or 4 or more times a day. In some embodiments, such a dose is providedtwice a week, once a week, twice a month or once a month. The amount maybe provided by oral consumption, infusion, or administered by othermeans familiar to those of skill in the art, such as transdermal ortransmucosal.

In other embodiments, the unit dose may be provided as an infusion. Forexample, the compositions described herein can be administeredintravenously, such as by an IV drip using IV solutions well known inthe art (e.g., isotonic saline (0.9% NaCl) or dextrose solution (e.g.,5% dextrose), optionally the intravenous solution further includespreservatives, e.g. sodium metabisulfite). For example, a dose can beprovided by infusion, such as by IV drip once a day, twice a week, oncea week, twice a month or once a month. Alternately, the unit dose isinfused once a day, twice a day, 3 times a day, or 4 or more times aday, for a period of time.

In other embodiments, the unit dose is from about 0.5 to about 500mg/kg, and all ranges within, of compound of the present application orpharmaceutically acceptable salt thereof.

In some embodiments, the unit dose is at least about 2 □g/kg to 500mg/kg, and all ranges within of a compound of the present application orpharmaceutically acceptable salt thereof.

Treatment

The present invention provides methods of 1) preventing, diagnosing,monitoring and/or treating a disorder in a subject in need thereof, 2)reducing one or more adverse effects associated with the treatment of adisorder and/or 3) increasing therapeutic efficacy in the treatment of adisorder.

In some embodiments, the method comprises, consists essentially of orconsists of preventing and/or treating a disorder in a subject in needthereof, comprising, consisting essentially of or consisting ofadministering to said subject a therapeutically effective amount of acomposition comprising, consisting essentially of or consisting of acompound of Formula I, II, or III.

In some embodiments, the method comprises, consists essentially of orconsists of preventing and/or treating a disorder in a subject in needthereof, comprising, consisting essentially of or consisting ofadministering to said subject a pharmaceutical composition comprising,consisting essentially of or consisting of a compound of Formula I, II,or III and a pharmaceutically acceptable carrier.

In some embodiments, the method comprises, consists essentially of orconsists of preventing, treating, and/or supplementing the treatment ofa disorder in a subject in need thereof, comprising, consistingessentially of or consisting of administering to said subject anutraceutical composition comprising, consisting essentially of orconsisting of a compound of Formula I, II, or III and an acceptablecarrier.

In some embodiments, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of a composition of the present invention. In some suchembodiments, the therapeutically effective amount comprises a preventioneffective amount. In some embodiments, the therapeutically effectiveamount comprises a treatment effective amount.

In some embodiments, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of a compound of Formula I:

wherein n is 2, 4, 6 or combinations thereof. In one variation themethod comprises administering a pharmaceutically acceptable salt orhydrate of said compound. In one embodiment of the compound of FormulaI, n is 2; in another embodiment, n is 4 and in yet another embodiment,n is 6. In another embodiment, the kit comprises one or more of thecompounds of Formula I, such as a kit comprising a compound where n is 2and a compound where n is 4, or a compound where n is 2 and a compoundwhere n is 6 or a compound where n is 4 and a compound where n is 6.Alternately, such a kit comprises a compound where n is 2 and a compoundwhere n is 4 and a compound where n is 6. In one variation of any aspector embodiment, the compound is in an isolated or purified form;alternately the compound is in a synthetic reaction mixture.

In another aspect, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of a compound of Formula II:

or a pharmaceutically acceptable salt or hydrate thereof wherein R is

and any diastereomers thereof. In one variation the compound is in anisolated or purified form; alternately the compound is in a syntheticreaction mixture.

In one variation, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form; alternatelythe compound is in a synthetic reaction mixture. In another variation,the method comprises, consists essentially of or consists ofadministering to said subject a therapeutically effective amount of acompound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form; alternatelythe compound is in a synthetic reaction mixture.

In one aspect, the method comprises, consists essentially of or consistsof administering to said subject a therapeutically effective amount of acompound of Formula III:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form; alternatelythe compound is in a synthetic reaction mixture.

In one aspect, the method comprises, consists essentially of or consistsof administering to said subject a therapeutically effective amount of acompound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form. In anothervariation, the compound is in a synthetic reaction mixture.

In another aspect, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of M14 or M15:

or a pharmaceutically acceptable salt or hydrate thereof. In onevariation, the compound is in an isolated or purified form. In anothervariation, the compound is in a synthetic reaction mixture.

In yet another aspect, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof. In oneembodiment, the method comprises administering one or more of M9, M9′and M9″ or a pharmaceutically acceptable salt or hydrate thereof. Inanother embodiment, the method comprises administering one or more ofM11, M11′ and M11″ or a pharmaceutically acceptable salt or hydratethereof. In one variation of any disclosed aspect or embodiment, themethod comprises administering a composition that does not include M2,M9 or M11. In another variation, the method comprises administering acomposition that comprises M2, M2′, M2″, M9, M9′, M9″, M11, M11′ andM11″ or a pharmaceutically acceptable salt. In yet another variation,the method comprises administering a composition that comprises M2′,M2″, M9′, M9″, M11′ and M11″. In one variation of any aspect orembodiment, the compound is in an isolated or purified form. In anothervariation, the compound is in a synthetic reaction mixture.

In some embodiments, the method comprises, consists essentially of orconsists of administering to said subject a therapeutically effectiveamount of a pharmaceutical composition of the present invention.

In some such embodiments, the composition is a compound of the presentinvention. In some such embodiments, the composition is a pharmaceuticalcomposition of the present invention. In other such embodiments, thecomposition is a nutraceutical composition of the present invention.

In some embodiments, administration of the composition results in theprevention and/or treatment of a first disorder and the preventionand/or treatment of a second disorder. For example, administration ofthe composition may result in the treatment of rectal cancer and theprevention of colon cancer (by preventing metastasis, for example).

In some embodiments, the subject exhibits one or more risk factorsassociated with the disorder. For example, the subject may have afamilial history of cancer, one or more pre-cancerous lesions,premalignant cells, preneoplastic cells or other aberrant phenotypesindicating probably progression to a cancerous state.

In some embodiments, the subject is a human.

Combination Therapy

In certain embodiments of the present application, the compounds of thepresent application can be used in a combination therapy with at leastone other therapeutic agent. The compounds and the therapeutic agent canact additively or, more preferably, synergistically.

In some embodiments, the other therapeutic agent is an antitumoralkylating agent, antitumor antimetabolite, antitumor antibiotics,plant-derived antitumor agent, antitumor organoplatinum compound,antitumor campthotecin derivative, antitumor tyrosine kinase inhibitor,monoclonal antibody, interferon, biological response modifier, hormonalanti-tumor agent, angiogenesis inhibitor, differentiating agent, or apharmaceutically acceptable salt thereof. In some embodiments, thecompound is administered in combination with surgery, radiation therapy,chemotherapy, gene therapy, RNA therapy, adjuvant therapy,immunotherapy, nanotherapy or a combination thereof. In someembodiments, the method further comprises administering to the subject atherapeutically effective amount of a pharmaceutical composition, thecomposition comprising a compound of the present application and apharmaceutically acceptable carrier. In some embodiments, the subject isa mammal. In some embodiments, the subject is a human.

Combination therapy includes the administration of a compound or salt ofthe present application and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).Combination therapy can be carried out either sequentially orsubstantially simultaneously. In the case of sequential administrationof more than one therapeutic agent, each therapeutic agent isadministered at a different time. In the case of simultaneousadministration, at least two of the therapeutic agents are administeredin a substantially simultaneous manner, either in the samepharmaceutical composition or in different pharmaceutical compositions.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. In one embodiment, a composition comprising acompound of the application is administered concurrently with theadministration of another therapeutic agent, which can be part of thesame composition as the compound of the application or a differentcomposition.

In another embodiment, a composition comprising a compound of theapplication is administered prior to, or subsequent to, administrationof another therapeutic agent. The therapeutic agents can be administeredin a variety of combinations. In some embodiments, the therapeuticagents are administered within about 1 minute, 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5hours, 5 hours, 5.5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1week, 2 weeks, 3 weeks or 4 weeks of one another, or any ranges therebetween.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected can be administered by intravenous injection while the othertherapeutic agents of the combination can be administered orally.Alternatively, for example, all therapeutic agents can be administeredorally or all therapeutic agents can be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

Combination therapy also encompasses the administration of the compoundas described above in further combination with other therapies includingbut not limited to chemotherapy, surgery, radiation therapy, genetherapy, immunotherapy, RNA therapy, adjuvant therapy, nanotherapy or acombination thereof. Where the combination therapy further comprises anon-drug treatment, the non-drug treatment can be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, by a significant period oftime. The compound and the other pharmacologically active agent can beadministered to a patient simultaneously, sequentially or incombination. It will be appreciated that when using a combination of theapplication, the compound of the application and the otherpharmacologically active agent can be in the same pharmaceuticallyacceptable carrier and therefore administered simultaneously. They canbe in separate pharmaceutical carriers such as conventional oral dosageforms which are taken simultaneously. The term “combination” furtherrefers to the case where the compounds are provided in separate dosageforms and are administered sequentially.

In some embodiments, treatment of cancer with a compound of the presentapplication is accompanied by administration of pharmaceutical agentsthat can alleviate the side effects produced by the antineoplasticagents.

EXAMPLES

The following examples are for illustrative purposes only and are notintended to be a detailed catalogue of all the different ways in whichthe present invention may be implemented or of all the features that maybe added to the present invention.

Starting materials useful for preparing compounds of the presentinvention and intermediates thereof are commercially available and/orcan be prepared by well-known synthetic methods. Other methods forsynthesis of the compounds described herein are either described in theart or will be readily apparent to the skilled artisan in view of thereferences provided above and can be used to synthesize conjugates ofthe application. One skilled in the art will therefore appreciate thatthe following Examples are exemplary and that numerous changes,modifications, and alterations can be employed without departing fromthe scope of the presently disclosed subject matter.

Experimental Part 1

Materials and Methods

[6]-Shogaol was purified from ginger extract using methods disclosed inSang S, et al., (2009) J Agric Food Chem 57:10645-10650. Sephadex LH-20,reverse-phase C18 silica gels, analytical and preparative thin-layerchromatography (TLC) plates (250- and 2000-□m thickness, 2-25-□mparticle size), and CDCl₃ were purchased from Sigma-Aldrich (St. Louis,Mo.). High-performance liquid chromatography (HPLC)-grade solvents andother reagents were obtained from VWR Scientific (South Plainfield,N.J.). All other chemicals were purchased from Sigma (St. Louis, Mo.) orThermo Fisher Scientific (Waltham, Mass.). Anhydrous reactions werecarried out in oven-dried glassware under a nitrogen atmosphere unlessotherwise noted. Analytical (250 μm thickness, 2-25 μm particle size)and preparative TLC plates (2000 μm thickness, 2-25 μm particle size)were purchased from Sigma (St. Louis, Mo.) and Sorbent Technologies(Atlanta, Ga.), respectively. Microsomes and NADPH-regenerating systemswere procured from BD Biosciences (Bedford, Mass.). 1-Aminobenzotriazole(ABT), 18β-glyccerhetinic acid (18β-GA), and2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) were purchased fromSigma Aldrich (St. Louis, Mo.). Liquid chromatography/mass spectrometry(LC/MS)-grade MeOH and water were obtained from Thermo Fisher Scientific(Waltham, Mass.). HCT-116 and HT-29 human colon cancer cells, H-1299human lung cancer cells, and CL-13 mouse lung cancer cells, CCD-18Cohuman fibroblast cells derived from colon, IMR-90 human diploidfibroblast cells derived from lung, and Eagle's minimum essential media(EMEM) were obtained from the American Type Culture Collection(Manassas, Va.). McCoy's 5A medium was purchased from Mediatech(Herndon, Va.) or Thermo Fisher Scientific (Waltham, Mass.). Fetalbovine serum (FBS) and penicillin/streptomycin were purchased fromGemini Bio-Products (West Sacramento, Calif.). MTT(3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide) wasprocured from Calbiochem-Novabiochem (San Diego, Calif.). Proteinase Kwas obtained from Ambion (Austin, Tex.). Apoptag Plus Peroxydase In SituApoptosis Detection Kit was purchased from Millipore Corporation(Billerica, Mass.). Glutathione, sulfatase from Aerobacter aerogenes,and β-glucuronidase from Helix aspersa were obtained from Sigma Aldrich(St. Louis, Minn.).

Nuclear Magnetic Resonance.

¹H (600 MHz), ¹³C (150 MHz), and all two-dimensional (2D) NMR spectrawere acquired on a Bruker AVANCE 600 MHz NMR spectrometer (Brucker,Inc., Silberstreifen, Rheinstetten, Germany). Compounds were analyzed inCDCl₃ or CD₃OD. Multiplicities are indicated by s (singlet), d(doublet), t (triplet), q (quartet), and br (broad). The ¹³C NMR spectraare proton decoupled.

HPLC Analysis.

An HPLC ESA electrochemical detector (ECD) (ESA, Chelmsford, Mass.)consisting of an ESA model 584 HPLC pump, an ESA model 542 autosampler,an ESA organizer, and an ESA ECD coupled with two ESA model 6210 foursensor cells was used. A Gemini C18 column (150×4.6 mm, 5 □m;Phenomenex, Torrance, Calif.) was used for chromatographic analysis at aflow rate of 1.0 ml/min. The mobile phases consisted of solvent A (30 mMsodium phosphate buffer containing 1.75% acetonitrile and 0.125%tetrahydrofuran, pH 3.35) and solvent B (15 mM sodium phosphate buffercontaining 58.5% acetonitrile and 12.5% tetrahydrofuran, pH 3.45). Thegradient elution had the following profile: 20% solvent B from 0 to 3min; 20 to 55% solvent B from 3 to 11 min; 55 to 60% solvent B from 11to 12 min; 60 to 65% solvent B from 12 to 13 min; 65 to 100% solvent Bfrom 13 to 40 min; 100% solvent B from 40 to 45 min; and then 20%solvent B from 45.1 to 50 min. The cells were then cleaned at apotential of 1000 mV for 1 min. The injection volume of the sample was10 □l. The eluent was monitored by the Coulochem electrode array system(ESA) with potential settings at −100, 0, 100, 200, 300, 400, and 500mV.

Waters preparative HPLC system (Waters, Milford, Mass.) with 2545 binarygradient module, Waters 2767 sample manager, Waters 2487autopurification flow cell, Waters fraction collector III, dual injectormodule, and a 2489 UV/visible detector was used to purify metabolites M6through M8 and M12. A Gemini-NX C18 column (250×30.0 mm i.d., 5 □m;Phenomenex) was used with a flow rate of 20.0 ml/min, and the separationwas performed with a mobile phase of MeOH/H2O. The gradient elution hadthe following profile: 70% solvent B from 0 to 30 min; 70 to 100%solvent B from 30 to 31 min; 100% solvent B from 31 to 36 min; 100 to70% solvent B from 36 to 37 min; and then 70% solvent B from 37 to 42min. The wavelength of the UV detector was set at 230 nm. Water andmethanol were used as mobile phases A and B, respectively.

Liquid Chromatography/Electrospray Ionization-Mass Spectrometry Method.

LC/MS analysis was performed with a Thermo-Finnigan Spectra

System, which consisted of an Accela high-speed mass spectrometry (MS)pump, an Accela refrigerated autosampler, and an LTQ Velos ion trap massdetector (Thermo Fisher Scientific) incorporated with heatedelectrospray ionization (H-ESI) interfaces. A Gemini C18 column (50×2.0mm i.d., 3 □m; Phenomenex) was used for separation at a flow rate of 0.2ml/min. The column was eluted from 100% solvent A (5% aqueous methanolwith 0.2% acetic acid) for 3 min, followed by linear increases insolvent B (95% aqueous methanol with 0.2% acetic acid) to 40% from 3 minto 15 min, to 85% from 15 to 45 min, to 100% from 45 to 50 min, and thenwith 100% solvent B from 50 to 55 min. The column was thenre-equilibrated with 100% solvent A for 5 min. The liquid chromatography(LC) eluent was introduced into the H-ESI interface.

The positive ion polarity mode was set for the H-ESI source with thevoltage on the H-ESI interface maintained at approximately 4.5 kV.Nitrogen gas was used as the sheath gas and auxiliary gas. Optimizedsource parameters, including ESI capillary temperature (300° C.),capillary voltage (50 V), ion spray voltage (3.6 kV), sheath gas flowrate (30 units), auxiliary gas flow rate (5 units), and tube lens (120V), were tuned using authentic [6]-shogaol. The collision-induceddissociation was conducted with an isolation width of 2 Da andnormalized collision energy of 35 for MS² and MS³. Default automatedgain control target ion values were used for MS, MS², and MS³ analyses.The mass range was measured from 50 to 1000 m/z. Data acquisition wasperformed with Xcalibur 2.0 version (Thermo Fisher Scientific).

Experimental Part 1

Treatment of Mice and Sample Collections.

Female C57BL/6J mice and female NJ mice were purchased from The JacksonLaboratory (Bar Harbor, Me.) and were allowed to acclimate for at least1 week before the start of the experiment. Mice were housed five percage and maintained in air-conditioned quarters with a room temperatureof 20±2° C., relative humidity of 50±10%, and an alternating 12-hlight/dark cycle. Mice were fed Purina Rodent Chow number 5001 (ResearchDiets; Purina, St. Louis, Mo.) and water and were allowed to eat anddrink ad libitum.

24-h urinary and fecal samples were collected using metabolic cages formetabolic profile analysis. In brief, [6]-shogaol in corn oil or cornoil only was administered to C57BL/6J mice by oral gavage (200 mg/kg).Fecal and urinary samples were collected in metabolic cages (five miceper cage) for 24 h after administration of vehicle (control group, n=5)or [6]-shogaol (treated group, n □ 5). In experiment 2, A/J mice wereadministrated [6]-shogaol by oral gavage (200 mg/kg per day) for 10days. Fecal samples were collected from mouse cages every 5 days. Thecombined fecal samples were used to purify the major metabolites of[6]-shogaol. These samples were stored at □80° C. before analysis. Inexperiment 3, A/J mice were treated with either 200 mg/kg [6]-shogaol incorn oil or corn oil only by oral gavage. Blood was collected fromanesthetized mice by cardiac puncture at 2 or 6 h after administrationof vehicle or [6]-shogaol (five mice per time point), and plasma wasisolated by centrifugation at 5000 rpm for 15 min in a refrigeratedcentrifuge. Plasma samples were then stored at −80° C. until analysis.

Fecal, Urinary, and Plasma Sample Preparation.

For acquisition of the metabolic profile, six pieces of each fecalsample (control and treated) were chosen and put into 2-ml tubes. Eachset was weighted (control, 128 mg; treated, 130 mg), and 1.2 ml ofMeOH/H2O (50/50)+0.1% acetic acid was added to each sample. Samples weresonicated for 90 min and then centrifuged at 17,000 rpm for 10 min. Thesupernatant (250 □l) was collected and diluted five times for analysis.Enzymatic deconjugation was performed as described previously withslight modifications (Shao et al., (2010) Rapid Commun Mass Spectrom24:1770-1778.). In brief, 250 □l of supernatant were dried under reducedpressure at 37° C., and the residue was resuspended in sodium phosphatebuffer (50 mM, pH 6.8). Samples were then treated with □-glucuronidase(250 units) and sulfatase (3 units) for 24 h at 37° C. and wereextracted twice with ethyl acetate. The ethyl acetate fraction was driedunder vacuum, and the solid was resuspended in 1.25 ml of 80% aqueousmethanol with 0.1% acetic acid for further analysis. For preparation ofthe urinary and plasma samples, 50 □l from each group (control group and[6]-shogaol treated group) were added to 1.2 ml of MeOH to precipitateproteins. After centrifugation at 17,000 rpm for 10 min, thesupernatants were transferred into vials for analysis. Enzymaticdeconjugation of the urinary and plasma samples was performed asdescribed above. In brief, 50 □l from each group (control group and[6]-shogaol-treated group) were treated with □-glucuronidase (250 units)and sulfatase (3 units) for 24 h at 37° C. and were extracted twice withethyl acetate. The ethyl acetate fraction was dried under vacuum, andthe solid was resuspended in 1.25 ml (for urine) or 250 □l (for plasma)of 80% aqueous methanol with 0.1% acetic acid for further analysis.

Purification of the Major Mouse Fecal Metabolites of [6]-Shogaol.

The mouse feces (228.29 g) collected as described above and wereextracted with MeOH/H₂O (50/50, 1000 ml each time) twice and then wereextracted with MeOH five times (1000 ml each time). The extract wasdried under reduced pressure at 37° C., and the residue (40.06 g) wasdissolved in water (800 ml) and partitioned successively with ethylacetate (5×500 ml) and 1-butanol (2×600 ml). The ethyl acetate-solubleportion (5.5 g) was subjected to a reversephase C18 column eluted with aMeOH/H₂O gradient system (3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1; v/v; 800 mlfor each gradient), giving 12 fractions. Fraction 7 was separated bypreparative HPLC to give fractions 7a and 7b. Fraction 7a wassuccessively separated on a preparative silica gel TLC plate (developedwith CHCl₃/MeOH, 100:1) and Sephadex LH-20 (eluted with EtOH) columnchromatography (CC) to give M11 (17 mg). Fraction 7b was purified on apreparative silica gel TLC plate (developed with CHCl₃/MeOH, 100:1) toyield two subfractions (7b1 and 7b2). Fraction 7b1 was first loaded on apreparative silica gel TLC plate (developed with n-hexane/EtOAc, 10:1)and then on Sephadex LH-20 (eluted with EtOH) CC to give M9 (0.5 mg) andM10 (0.8 mg). Fraction 7b2 was subjected to preparative HPLC to give M12(0.6 mg). Fraction 8 was loaded on a preparative silica gel TLC plate(developed with n-hexane/EtOAc, 10:1) to give fractions 8a through 8c.Fraction 8a was subjected to preparative HPLC to give one majorfraction, which was then successively separated on a preparative silicagel TLC plate (developed with CHCl₃/MeOH, 100:1) and Sephadex LH-20(eluted with EtOH) CC to give M6 (0.5 mg). Fraction 8b was subjected toSephadex LH-20 (eluted with EtOH) CC and then preparative HPLC to giveM7 (0.5 mg). Fraction 8c was first loaded on a preparative silica gelTLC plate (developed with n-hexane/EtOAc, 10:1) and then on preparativeHPLC to give M8 (4.0 mg). ¹H and ¹³C NMR data of M6 through M12 arelisted in Tables 1 and 2.

TABLE 1 ¹H and ¹³C NMR spectroscopic data of M6 through M9 and M11M6^(a) M7^(b) M8^(b) M9^(b) M11^(b) No. δ_(H) multi (J in Hz) δ_(C)δ_(H) multi (J in Hz) δ_(C) δ_(H) multi (J in Hz) δ_(C) δ_(H) multi (Jin Hz) δ_(C) δ_(H) multi (J in Hz) δ_(C)  1′ 133.6 133.0 134.1 134.0133.1  2′ 6.77 br s 111.6 6.71 d (1.2) 111.0 6.72 br s 115.4 6.71 d(1.5) 111.0 6.70 br s 111.0  3′ 147.4 146.3 143.6 146.4 146.4  4′ 144.0143.9 141.9 143.7 143.9  5′ 6.70 br d (7.8) 114.6 6.83 d (7.8) 114.36.78 d (7.8) 115.4 6.84 d (7.9) 114.2 6.82 br d (7.9) 114.3  6′ 6.63 brd (7.8) 128.3 6.69 dd (7.8, 1.2) 120.8 6.61 d (7.8) 120.5 6.69 dd (8.0,1.5) 120.9 6.67 br d (7.9) 120.8  1 2.58 m 31.1 2.85 t (7.8) 29.2 2.79 t(7.2) 29.2^(c) 2.74 m; 2.62 m 31.7 2.84 t (7.5) 29.5  2 1.73 39.3 2.76 m45.8 2.70 43.4 1.78 m; 1.71 m 39.4 2.70 t (7.5) 44.6  3 3.99 m 71.6209.0 211.5 3.64 m 71.4 210.6  4 5.47 dd (15.4, 7.1) 131.3 2.66 dd(16.2, 7.8); 47.6 2.38 t (7.4) 43.2 1.48 m 37.6 2.38 t (7.4) 43.1 2.42dd (16.2, 4.8)  5 5.64 dt (15.4, 7.1) 132.9 3.67 m 77.1 1.56 m 23.8 1.30m 29.4 1.56 m 23.8  6 2.06 m 31.9 1.49 m; 1.43 m 33.8 1.31 m 29.0^(c)1.30 m 29.4 1.26 m 29.0  7 1.29 m 28.8 1.31 m 24.7 1.31 m 29.1^(c) 1.44m; 1.32 m 25.6 1.26 m 29.0  8 1.31 m 31.2 1.31 m 31.9 1.31 m 31.6 1.28 m31.7 1.26 m 31.6  9 1.34 m 22.2 1.31 m 22.6 1.31 m 22.6 1.30 22.6 1.29 m22.5 10 0.92 t (7.2) 13.1 0.90 t (7.2) 13.9 0.89 t (7.2) 14.1 0.91 t(7.1) 14.0 0.89 t (7.1) 14.0  3′-OMe 3.84 s 54.8 3.89 s 55.9 3.90 s 55.93.57 s 55.9  5-OMe 3.30 s 56.9  4′-OH 5.47 s 5.47 s ^(a)Data weremeasured in CD₃OD at 600 (²H) and 150 MHz (¹³C). ^(b)Data were measuredin CDCl₃ at 600 (¹H) and 150 MHz (¹³C). Chemical shifts (δ) are in ppmbeing relative to CD₃OD and CDCl₃ ^(c)Data can be exchanged with eachother.

TABLE 2 ¹H and ¹³C NMR spectroscopic data of [6]-shogaol, M10, M12 andsynthetic 5-N-acetylcysteinyl-[6]-shogaol [6]-Shogaol^(a) M10^(b)M12^(b) 5-N-acetylcysteinyl-[6]-shogaol^(a) No. δ_(H) multi (J in Hz)δ_(C) δ_(H) multi (J in Hz) δ_(C) δ_(H) multi (J in Hz) δ_(C) δ_(H)multi (J in Hz) δ_(C)  1′ 132.6 132.9 134.0 134.0  2′ 6.80 d (1.8) 111.86.71 d (1.5) 111.0 6.73 d (2.2) 111.0 6.81 d (1.8) 111.7  3′ 147.6 146.4146.4 149.0  4′ 144.5 143.9 143.7 145.9  5′ 6.71 d (8.0) 114.7 6.84 d(8.0) 114.3 6.84 d (7.9) 114.2 6.71 d (8.0) 114.6  6′ 6.63 dd (8.0, 1.8)120.4 6.69 dd (8.0, 1.5) 120.8 6.71 dd (7.9, 2.2) 120.9 6.65 dd (8.0,1.8) 120.2  1 2.88 m 31.1 2.86 t (7.5) 29.3 2.64 m; 2.75 m 31.7 2.80 m27.5  2 2.66 m 41.3 2.75 m 45.5 1.79 m; 1.74 m 39.8 2.74 dd (17.1, 6.3);49.7 2.67 dd (17.1, 6.3)  3 201.5 208.4 4.00 m 68.9 211.2  4 6.12 br d(15.9) 130.0 2.59 dd (16.6, 6.5); 48.5 1.71 m; 1.62 m 40.6 2.74 m 46.12.69 dd (16.6, 6.5)  5 6.90 dt (15.9, 7.02) 148.7 3.04 m 41.6 2.75 m43.2 3.14 m 42.6  6 2.22 m 32.1 1.50 m 34.4 1.61 m 34.8 1.51 m 33.7  71.48 m 27.6 1.42 m; 1.38 m 26.5 1.44 m 26.6 1.34 m 26.6  8 1.34 m 29.81.27 m 31.6 1.31 m 31.9 1.27 m 30.9  9 1.34 m 22.1 1.31 m 22.6 1.31 m22.6 1.33 m 22.2 10 0.93 t (7.1) 13.0 0.90 t (7.1) 14.0 0.91 t (7.1)14.1 0.92 14.4  3′-OMe 3.84 s 3.89 s 55.9 3.90 s 55.7 56.4  5-SMe 2.04 s13.3 2.05 12.3  4′-OH 5.45 s 5.47 s  3-OH 2.25 d (4.9)  1′′ 3.00 dd(4.7, 13.7); 30.4 2.92 dd (7.1, 13.7)  2′′ 4.58 dd (7.1, 4.7) 54.6  3′′173.1  4′′ 173.1  5′′ 2.01 s 22.6 ^(a)Data were measured in CD₃OD at 600(¹H) and 150 MHz (¹³C). ^(b)Data were measured in CDCl₃ at 600 (²H) and150 MHz (¹³C). Chemical shifts (δ) are in ppm being relative to CD₃ODand CDCl₃.

Synthesis of 5-N-Acetylcysteinyl-[6]-Shogaol

[6]-Shogaol (235 mg, 0.8 mmol) was dissolved in ethanol (40 ml) andadded dropwise to a solution of N-acetylcysteine[1076 mg, 6.6 mmol in100 ml of phosphate buffered saline (PBS) at pH 7.4] at 37° C. Afterstirring for 24 h, the reaction mixture was extracted with ethylacetate. The organic phase was then separated and dried, and the residue(520 mg) was redissolved in MeOH. The reconstituted solution wassubjected to a reverse-phase C18 column and was eluted with a mobilephase of MeOH/H₂O (70:30, v/v) at a flow rate of 2 ml/min. The sampleswere combined on the basis of the TLC analysis and were dried to obtain240 mg (yield 64%) of final product. ¹H and ¹³C NMR data of5-N-acetylcysteinyl-[6]-shogaol are listed in Table 2.

Metabolism of [6]-Shogaol in Cancer Cells.

Cells (1.0×10⁶) were plated in six-well culture plates and were allowedto attach for 24 h at 37° C. in 5% CO₂ incubator. [6]-Shogaol [indimethyl sulfoxide (DMSO)] was added to McCoy's 5A medium (containing10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine) toreach a final concentration of 10 □M and was incubated with differentcancer cell lines (HCT-116, HT-29, H-1299, and CL-13). At different timepoints (0, 30 min, 1, 2, 4, 6, 8, and 24 h), samples of supernatant weretaken and transferred to vials containing 10 □l of 0.2% ascorbic acid tostabilize [6]-shogaol and its metabolites. The metabolites wereextracted from media by addition of equal volume of acetonitrile andcentrifugation, in which the supernatant was harvested. The samples werethen diluted 5-fold in acetonitrile and were analyzed by HPLC ECD.

Growth Inhibition of Human Cancer Cells.

Cell growth inhibition was determined by a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)colorimetric assay (Mosmann T (1983) J Immunol Methods 65:55-63.). Humancolon cancer (HCT-116) and human lung cancer (H-1299) cells (3000cells/well) were plated in 96-well microtiter plates and were allowed toattach for 24 h at 37° C. The test compounds (in DMSO) were added tocell culture medium to desired final concentrations (0-80 □M; final DMSOconcentrations for control and treatments were 0.1%). After the cellswere cultured for 24 h, the medium was aspirated, and the cells weretreated with 200 □l of fresh medium containing 2.41 mM MTT. Afterincubation for 3 h at 37° C., the medium containing MTT was aspirated,100 of DMSO was added to solubilize the formazan precipitate, and theplates were shaken gently for an hour at room temperature.

Absorbance values were derived from the plate reading at 550 nm on amicrotiter plate reader. The reading reflected the number of viablecells and was expressed as a percentage of viable cells in the control.Both HCT-116 and H-1299 cells were cultured in McCoy's 5A medium. All ofthe above media were supplemented with 10% fetal bovine serum, 1%penicillin/streptomycin, and 1% glutamine, and the cells were kept in a37° C. incubator with 95% humidity and 5% CO₂.

Terminal Deoxynucleotidyl Transferase Deoxyuridine Triphosphate Nick-EndLabeling Assay.

HCT-116 and H-1299 cells were seeded in six-well plates at 1.0×10⁵cells/well and were incubated at 37° C. in a 5% CO₂ incubator. After 24h, fresh media supplemented with DMSO (control), [6]-shogaol (10 or 20□M), M9 (40 or 80 □M), or M11 (40 or 80 □M) were added to the wells.After 24-h incubation, cells were washed and pretreated for 15 min atroom temperature with a solution of 20 □g/ml proteinase K. Cells werethen washed twice with PBS pH 7.4 and were fixed for 10 min at roomtemperature using 10% neutral formaldehyde solution. After two washes indistilled H₂O, cells were resuspended in 100 □l of distilled H₂O andwere applied on silanized microscope slides. Slides were incubatedovernight at 37° C. and were washed twice with PBS. Terminaldeoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling(TUNEL) assay was then performed according to the manufacturer'sprotocol. Cells were observed under 400× power using a Zeiss A1microscope (Carl Zeiss, Inc., Thornwood, N.Y.).

Ten fields per slide were evaluated, and TUNEL-positive cells (withbrown coloration in the nucleus) were expressed as a percentage of thetotal number of cells contained in a field. Statistical Analysis. Forsimple comparisons between two groups, twotailed Student's t test wasused. A p value of less than 0.05 was considered statisticallysignificant in all the tests.

Experimental Part 2

General Procedure A for Michael Addition Reaction.

A catalyst amount of NaHCO₃ (0.05 eq) was added to a mixture of[6]-shogaol (1.0 eq) and amino acid (3.0 eq) in methanol/water (1:1,v/v). The mixture was stirred at room temperature (rt) for 3-48 h,adjusted pH until 6 with a diluted HOAc solution (0.1 M), and extractedwith n-butanol (BuOH) (5 mL×3). Combined organic layers wereconcentrated under reduced pressure at 20° C. The residue was subjectedto column chromatography (CC) on Sephadex LH-20, and eluted with 90%ethanol in water, producing the desired thiol conjugates M2, M5, or M13.

General Procedure B for the Synthesis of Ketone Reduced MetabolitesUsing NaBH₄.

NaBH₄ (2.5-4.0 eq) was added to a solution of M2, M5 or [6]-shogaol (1.0eq) in methanol at 0° C. After stirring at 0° C. for 2 h, the reactionmedia was neutralized with a diluted HOAc solution (0.1 M) and extractedwith n-BuOH (5 mL×3). Combined organic layers were concentrated underreduced pressure. The residue was purified by CC on Sephadex LH-20 orpreparative TLC to produce the required compounds M1, M4, or M9.

Synthesis of 5-Cysteinyl-[6]-Shogaol (M2)

General procedure A was followed using [6]-shogaol (200 mg, 0.72 mmol),L-cysteine (263 mg, 2.17 mmol), and NaHCO₃ (3 mg, 0.036 mmol) inmethanol/water (10 mL, 1:1, v/v). The mixture was stirred at rt for 24h. The final residue was purified by CC on Sephadex LH-20 with 90%ethanol in water to give the title compound M2 as a white solid (170 mg,yield 60%). M2 (a mixture of diastereomers): ¹H NMR (600 MHz, CD₃OD) δ6.77 (1H, d, J=1.5 Hz, H-2′), 6.67 (1H, d, J=8.0 Hz, H-5′), 6.61 (1H,dd, J=8.0, 1.5 Hz, H-6′), 2.77 (2H, m, H-1), 2.74 (2H, m, H-2), 2.71(1H, m, H-4a), 2.63 (1H, m, H-4b), 3.12 (1H, m, Hminor-5) and 3.08 (1H,m, Hmajor-5), 1.53 (2H, m, H-6), 1.39 (2H, m, H 7), 1.28 (2H, m, H-8),1.33 (2H, m, H-9), 0.89 (3H, t, J=7.0 Hz, H-10), 3.82 (3H, s, OMe-3′),3.62 (1H, dd, J=9.3, 3.7 Hz, HCys-α, major) and 3.59 (1H, dd, J=9.3, 3.7Hz, HCys-α, minor), 3.18 (1H, dd, J=14.5, 3.7 Hz, HCys-βa), and 2.84(1H, dd, J=14.5, 9.3 Hz, HCys-βb); 13C NMR (150 MHz, CD3OD) δ 133.8 (s,C-1′), 113.1 (d, C-2′), 148.9 (s, C-3′), 145.8 (s, C-4′), 116.2 (d,C-5′), 121.7 (d, C-6′), 30.3 (t, C-1), 47.5 (t, C-2), 211.8 (s, C═O,C-3), 49.6 (t, C-4), 42.3 (2d, C-5), 36.8 (2t, C-6), 27.4 (2t, C-7),32.6 (2t, C-8), 23.6 (t, C-9), 14.4 (2q, C-10), 56.4 (q, OMe-3′), 56.3(d, CCys-α), 32.8 (2t, CCys-β), and 172.5 (s, Cys α-COOH); positiveAPCIMS: m/z 398 [M+H]⁺.

Synthesis of 5-cysteinyl-M6 (M1)

General procedure B was followed using M2 (74 mg, 0.19 mmol) and NaBH₄(28 mg, 0.75 mmol) in methanol (3 mL). The resulting solution wasextracted with n-BuOH (5 mL×3). Combined organic layers were evaporatedunder reduced pressure at 20° C. The final residue was purified by CC onSephadex LH-20 with 90% ethanol in water to give the title compound M1as a white solid (68 mg, yield 90%); M1 (a mixture of diastereomers): ¹HNMR (600 MHz, CD₃OD) δ 6.77 (1H, d, J=1.7 Hz, H-2′), 6.69 (1H, d, J=8.0Hz, H-5′), 6.62 (1H, dd, J=8.0, 1.7 Hz, H-6′), 2.68 (1H, m, H-1a), 2.58(1H, m, H-1b), 1.72 (2H, m, H-2), 3.90 (1H, m, Hminor-3) and 3.66 (1H,m, Hmajor-3), 1.71 (2H, m, H-4), 2.94 (1H, m, H-5), 1.66 (1H, m, H-6a),1.52 (1H, m, H-6b), 1.44 (2H, m, H-7), 1.28 (2H, m, H-8), 1.33 (2H, m,H-9), 0.89 (3H, t, J=7.0 Hz, H-10), 3.82 (3H, s, OMe-3′), 3.64 (1H, m,HCys-α), 3.15 (1H, m, HCys-βa), and 2.85 (1H, m, HCys-βb); 13C NMR (150MHz, CD3OD) δ 135.1 (s, C-1′), 113.2 (d, C-2′), 148.8 (s, C-3′), 145.5(s, C-4′), 116.1 (d, C-5′), 121.8 (d, C-6′), 32.5 (2t, C-1), 41.1 (2t,C-2), 69.3 (2d, C-3), 43.9 (t, C-4), 43.8 (2d, C-5), 35.0 (2t, C-6),27.1 (2t, C-7), 33.0 (t, C-8), 23.6 (t, C-9), 14.4 (q, C-10), 56.4 (q,OMe-3′), 55.8 (2d, CCys-α), 32.8 (4t, CCys-β), and 172.8 (s, Cysα-COOH); positive APCIMS: m/z 400 [M+H]⁺.

Synthesis of 5-N-acetylcysteinyl-[6]-shogaol (M5)

General procedure A was followed using [6]-shogaol (200 mg, 0.72 mmol),N-acetyl-L-cysteine (354 mg, 2.17 mmol), and NaHCO₃ (3 mg, 0.036 mmol)in methanol/water (10 mL, 1:1, v/v). The mixture was stirred at rt for72 h. The final residue was purified by CC on Sephadex LH-20 with 90%ethanol in water to give title compound M5 as a white solid (252 mg,yield 80%); M5 (a mixture of diastereomers): ¹H NMR (600 MHz, CD₃OD)6.77 (1H, d, J=1.7 Hz, H-2′), δ 6.67 (1H, d, J=8.0 Hz, H-5′), 6.61 (1H,dd, J=8.0, 1.7 Hz, H-6′), 2.78 (2H, m, H-1), 2.77 (2H, m, H-2), 2.70(1H, dd, J=16.8, 8.1 Hz, H-4a), 2.64 (1H, dd, J=16.8, 6.3 Hz, H-4b),3.10 (1H, m, H-5), 1.48 (2H, m, H-6), 1.38 (2H, m, H-7), 1.25 (2H, m,H-8), 1.28 (2H, m, H-9), 0.89 (3H, t, J=7.0 Hz, H-10), 3.82 (3H, s,OMe-3′), 4.58 (1H, dd, J=8.1, 4.8 Hz, HCys-α, major) and 4.53 (1H, dd,J=8.1, 4.8 Hz, HCys-α, minor), 3.02 (1H, dd, J=13.6, 4.8 Hz, HCys-βa,minor) and 2.96 (1H, dd, J=13.6, 4.8 Hz, HCys-βa, major), 2.89 (1H, dd,J=13.6, 7.2 Hz, HCys-βb, major) and 2.76 (1H, dd, J=13.6, 7.2 Hz,HCys-βb, minor), and 2.01 (3H, s, CH3CO, major) and 1.98 (3H, s, CH3CO,minor); positive APCIMS: m/z 440 [M+H]⁺.

Synthesis of 5-N-acetylcysteinyl-M6 (M4)

General procedure B was followed using M5 (151 mg, 0.34 mmol) and NaBH₄(53 mg, 1.38 mmol) in methanol (10 mL). The resulting solution wasextracted with n-BuOH (10 mL×3). Combined organic layers were evaporatedunder reduced pressure at 20° C. The final residue was purified by CC onSephadex LH-20 with 90% ethanol in water to give the title compound M4as a white solid (100 mg, yield 66%); M4 (a mixture of diastereomers):¹H NMR (600 MHz, CD₃OD) δ 6.77 (1H, brs, H-2′), 6.68 (1H, d, J=8.0 Hz,H-5′), 6.62 (1H, dd, J=8.0 Hz, H-6′), 2.67 (1H, m, H-1a), 2.58 (1H, m,H-1b), 1.72 (2H, m, H-2), 3.88 (1H, m, Hminor-3) and 3.70 (1H, m,Hmajor-3), 1.68 (2H, m, H-4), 2.83 (1H, m, H-5), 1.60 (1H, m, H-6a),1.44 (1H, m, H-6b), 1.45 (2H, m, H-7), 1.28 (2H, m, H-8), 1.33 (2H, m, H9), 0.89 (3H, t, J=7.0 Hz, H-10), 3.83 (3H, s, OMe-3′), 4.54 (1H, m,HCys-α), 3.00 (1H, m, HCys-βa), 2.80 (1H, m, HCys-βb), and 1.98 (3H, s,CH3CO); 13C NMR (150 MHz, CD3OD) δ 135.1 (2s, C-1′), 113.2 (d, C-2′),148.8 (s, C-3′), 145.5 (s, C-4′), 116.1 (d, C-5′), 121.8 (d, C-6′), 32.5(t, C-1), 41.0 (2t, C-2), 69.3 (4d, C-3), 44.3 (4t, C-4), 43.8 (2d,C-5), 35.2 (t, C-6), 27.2 (4t, C-7), 32.9 (t, C-8), 23.6 (t, C-9), 14.4(2q, C-10), 56.4 (q, OMe-3′), 54.2 (2d, CCys-α), 32.7 (2t, CCys-β),173.2 (s, Cys α-COOH), 174.0 (s, CH3CO), and 22.4 (q, CH3CO); positiveAPCIMS: m/z 442 [M+H]⁺.

Synthesis of 1-(4′-hydroxy-3′-methoxyphenyl)-4-decen-3-ol (M6)

A solution of [6]-shogaol (138 mg, 0.5 mmol) in methanol (10 mL) wascooled to −78° C., CeCl₃.7H₂O (745 mg, 2.0 mmol) was added and themixture was stirred at −78° C. for 10 min. Then, NaBH4 (48 mg, 1.25mmol) was added to the mixture and allowed to react at −78° C. for 30min. The reaction was quenched by saturated aqueous NH₄Cl solution (20mL) and extracted with ethyl acetate (20 mL×3). The organic phases wereseparated, pooled, washed with water (10 mL×2) and brine (10 mL×1),dried over Na₂SO₄, and evaporated in vacuo. The residue was subjected topreparative TLC (hexane/EtOAc=3:1) to produce the title compound M6 as acolorless oil (139 mg, yield 100%); ¹H NMR (600 MHz, CDCl₃) δ 6.70 (1H,d, J=1.7 Hz, H-2′), 6.82 (1H, d, J=8.0 Hz, H-5′), 6.68 (1H, dd, J=8.0,1.7 Hz, H-6′), 2.62 (2H, m, H-1), 1.85 (1H, m, H-2a), 1.78 (1H, m,H-2b), 4.07 (1H, m, H-3), 5.49 (1H, dd, J=15.3, 6.7 Hz, H-4), 5.65 (1H,dt, J=15.3, 6.7 Hz, H-5), 2.03 (2H, m, H-6), 1.38 (2H, m, H-7),1.32-1.25 (4H, m, H-8 and H-9), 0.89 (3H, t, J=6.9 Hz, H-10), and 3.87(3H, s, OMe-3′); positive APCIMS: m/z 279 [M+H]⁺.

Synthesis of 5-methoxy-1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-one (M7)

A solution of [6]-shogaol (100 mg, 0.36 mmol) in methanol (5 mL) at 0°C. was treated with a solution of sodium (21 mg, 0.91 mmol) in methanol(1 mL). After 4.0 h, glacial acetic acid (0.5 mL) was added, and thesolution was concentrated under reduced pressure. The residue wasdissolved in water (5 mL), and extracted with ethyl acetate (5 mL×3).The organic phases were pooled, washed with water (5 mL×2) and brine (5mL×1), dried over Na₂SO₄, and evaporated in vacuo. The residue wassubjected to preparative TLC (hexane/EtOAc=4:1) to give the titlecompound M7 as a yellow oil (100 mg, yield 90%); ¹H NMR (600 MHz, CDCl₃)δ 6.69 (1H, d, J=1.6 Hz, H-2′), 6.82 (1H, d, J=8.1 Hz, H-5′), 6.66 (1H,dd, J=8.1, 1.6 Hz, H-6′), 2.75 (2H, m, H-1), 2.83 (2H, t, J=7.5 Hz,H-2), 2.64 (1H, dd, J=15.7, 7.6 Hz, H-4a), 2.40 (1H, dd, J=15.7, 4.7 Hz,H-4b), 3.66 (1H, m, H-5), 1.48 (1H, m, H-6a), 1.42 (1H, m, H-6b),1.31-1.25 (6H, m, ranged from H-7 to H-9), 0.88 (3H, t, J=7.1 Hz, H-10),and 3.87 (3H, s, OMe-3′); positive APCIMS, m/z 309 [M+H]⁺.

Synthesis of 5-methylthio-1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-one(M10)

A solution of NaSCH₃ in water (15% w/w, 1.5 mL, 3.19 mmol) was added toa solution of [6]-shogaol (100 mg, 0.36 mmol) in THF (10 mL) at rt inportions. After stirring for 6.0 h, 10 mL of water was added, followedby extraction with ethyl acetate (10 mL×3). The organic phases wereseparated, pooled, washed with water (10 mL×2) and brine (10 mL×1),dried over Na₂SO₄, and evaporated in vacuo. The residue was loaded topreparative HPLC (methanol in water: 70%-100% in 50 min) to give thetitle compound M10 as a yellow oil (70 mg, yield 60%); ¹H NMR (600 MHz,CDCl₃) δ 6.69 (1H, d, J=1.6 Hz, H-2′), 6.82 (1H, d, J=8.0 Hz, H-5′),6.67 (1H, dd, J=8.0, 1.6 Hz, H-6′), 2.73 (2H, m, H-1), 2.84 (2H, t,J=7.6 Hz, H-2), 2.67 (1H, dd, J=16.6, 7.5 Hz, H-4a), 2.57 (1H, dd,J=16.6, 6.4 Hz, H-4b), 3.02 (1H, m, H-5), 1.49 (2H, m, H-6), 1.42 (1H,m, H-7a), 1.36 (1H, m, H-7b), 1.32-1.24 (4H, m, H-8 and H-9), 0.88 (3H,t, J=6.9 Hz, H-10), 3.87 (3H, s, OMe-3′), and 2.03 (3H, s, SCH3-5);positive APCIMS: m/z 325 [M+H]⁺.

Synthesis of 1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-one (M11)

A solution of [6]-shogaol (276 mg, 1.0 mmol) in THF (2 mL) at rt wastreated with 10% Pd/C (30 mg, 10% w/w) under H₂. The mixture was stirredat rt overnight and filtered. The filtrate was concentrated underreduced pressure. The residue was loaded to preparative TLC(hexane/EtOAc=8:1) to give the title compound M11 as a yellow oil (272mg, yield 98%); ¹H NMR (600 MHz, CDCl₃) δ 6.69 (1H, d, J=1.6 Hz, H-2′),6.82 (1H, d, J=8.0 Hz, H-5′), 6.66 (1H, dd, J=8.0, 1.7 Hz, H-6′), 2.69(2H, t, J=7.4 Hz, H-1), 2.82 (2H, t, J 7.4 Hz, H-2), 2.37 (1H, t, J=7.4Hz, H-4), 1.54 (2H, m, H-5), 1.30-1.24 (8H, m, ranged from H-6 to H-9),0.87 (3H, t, J=6.8 Hz, H-10), and 3.87 (3H, s, OMe-3′); positive APCIMS,m/z 279 [M+H]⁺.

Synthesis of 1-(3′,4′-dihydroxyphenyl)-decan-3-one (M8)

A solution of BBr₃ in dichloromethane (DCM) (1.0 M, 0.67 mL, 0.67 mmol)was added dropwise to a solution of M11 (74 mg, 0.27 mmol) in DCM (3 mL)at −78° C. The reaction was allowed to warm up to rt for 2.0 h, quenchedwith water (10 mL), and extracted with ethyl acetate (10 mL×3). Theorganic phases were separated, pooled, washed with water (10 mL×2) andbrine (10 mL×1), dried over Na₂SO₄, and evaporated in vacuo. The residuewas subjected to preparative TLC (DCM/Methanol=20:1) to give the titlecompound M8 as a red solid (50 mg, yield 70%); ¹H NMR (600 MHz, CDCl₃) δ6.70 (1H, d, J=1.9 Hz, H-2′), 6.76 (1H, d, J=8.0 Hz, H-5′), 6.59 (1H,dd, J=8.0, 1.9 Hz, H-6′), 2.69 (2H, t, J=7.4 Hz, H-1), 2.78 (2H, t,J=7.4 Hz, H-2), 2.37 (2H, t, J=7.4 Hz, H-4), 1.54 (2H, m, H-5),1.30-1.24 (8H, m, ranged from H-6 to H-9), and 0.87 (3H, t, J=6.8 Hz,H-10); positive APCIMS: m/z 265 [M+H]⁺.

Synthesis of 1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-ol (M9)

General procedure B was followed using M11 (100 mg, 0.36 mmol) and NaBH₄(34 mg, 0.90 mmol) in methanol (2 mL). The resulting solution wasextracted with ethyl acetate (5 mL×3). Combined organic layers wereconcentrated under reduced pressure. The final residue was purified bypreparative TLC (DCM/Methanol=40:1) to produce the title compound M9 asa white solid (90 mg, yield 90%); ¹H NMR (600 MHz, CDCl₃) δ 6.71 (1H, d,J=1.6 Hz, H-2′), 6.83 (1H, d, J=8.0 Hz, H-5′), 6.69 (1H, dd, J=8.0, 1.6Hz, H-6′), 2.72 (1H, m, H-1a), 2.60 (1H, m, H-1b), 1.76 (1H, m, H-2a),1.71 (1H, m, H-2b), 3.62 (1H, m, H-3), 1.48 (2H, m, H-4), 1.44 (2H, m,H-5), 1.32-1.26 (8H, m, ranged from H-6 to H-9), 0.88 (3H, t, J=6.8 Hz,H-10), and 3.88 (3H, s, OMe-3′); positive APCIMS: m/z 281 [M+H]⁺.

Synthesis of 5-methylthio-1-(4′-Hydroxy-3′-methoxyphenyl)-decan-3-ol(M12)

General procedure B was followed using M10 (39 mg, 0.12 mmol) and NaBH₄(11 mg, 0.30 mmol) in methanol (3 mL). The resulting solution wasextracted with ethyl acetate (5 mL×3). The combined organic layers wereconcentrated under reduced pressure. The final residue was purified bypreparative TLC (DCM/Methanol=50:1) to produce the title compound M12 asa yellow oil (39 mg, yield 100%); Mixture of diastereomers: ¹H NMR (600MHz, CDCl₃) δ 6.71 (1H, d, J=1.5 Hz, H-2′), 6.82 (1H, d, J=8.0 Hz,H-5′), 6.68 (1H, dd, J=8.0, 1.5 Hz, H-6′), 2.74 (1H, m, H-1a), 2.61 (1H,m, H-1b), 1.75 (2H, m, H-2), 3.98 (1H, m, Hminor-3) and 3.80 (1H, m,Hmajor-3), 1.70 (1H, m, H-4a), 1.65 (1H, m, H-4b), 2.75 (1H, m, H-5),1.61 (2H, m, H-6), 1.44 (2H, m, H-7), 1.32-1.23 (4H, m, H-8 and H-9),0.88 (3H, t, J=7.0 Hz, H-10), 3.86 (3H, s, OMe-3′), and 2.02 (3H, s,SMe-5); positive APCIMS, m/z 327 [M+H]⁺.

Synthesis of 5-glutathiol-[6]-shogaol (M13)

General procedure A was followed using [6]-shogaol (100 mg, 0.36 mmol),reduced L-glutathione (333 mg, 1.09 mmol), and NaHCO₃ (1.5 mg, 0.018mmol) in methanol/water (8 mL, 1:1, v/v). The mixture was stirred at rtfor 3 h. The final residue was purified by CC on Sephadex with 90%ethanol in water to give the title compound M13 as a white solid (168mg, yield 80%); M13 (a mixture of diastereomers): ¹H NMR (600 MHz,CD₃OD) δ 6.77 (1H, d, J=1.6 Hz, H-2′), 6.68 (1H, d, J=8.0 Hz, H-5′),6.61 (1H, dd, J=8.0, 1.6 Hz, H-6′), 2.78-2.75 (4H, m, H-1 and H-2),2.74-2.61 (2H, m, H-4), 3.10 (1H, m, H-5), 1.51-1.45 (2H, m, H-6),1.42-1.33 (2H, m, H-7), 1.25 (2H, m, H-8), 1.28 (2H, m, H-9), 0.88 (3H,t, J=7.3 Hz, H-10), 3.82 (3H, s, OMe-3′), 3.65 (1H, m, HGlu-α), 2.13(2H, m, HGlu-13), 2.55 (1H, m, HGlu-γa), 2.51 (1H, m, HGlu-γb), 4.50(1H, dd, J=8.5, 5.1 Hz, HCys-α), 3.05-2.95 (1H, m, HCys-βa), 2.84-2.80(1H, m, HCys-βb), and 3.80 (2H, brs, HGly-α); 13C NMR (150 MHz, CD3OD) δ133.9 (s, C-1′), 113.2 (d, C-2′), 148.9 (s, C-3°), 145.7 (s, C-4′),116.2 (d, C-5′), 121.8 (d, C-6′), 30.4 (t, C-1), 46.0 (2t, C-2), 211.2(s, C═O, C-3), 49.8 (2t, C-4), 42.2 (2d, C-5), 36.2 (2t, C-6), 27.5 (2t,C-7), 32.8 (t, C-8), 23.6 (t, C-9), 14.4 (q, C-10), 56.4 (q, OMe-3′),55.4 (d, CGlu-α), 27.8 (t, CGlu-β), 33.0 (t, CGlu-γ), 174.0 (s, Gluα-COOH), 175.2 (s, Glu γ-CON), 55.0 (2d, CCys-α), 33.3 (2t, CCys-β),172.9 (s, Cys α-CON), 45.0 (t, CGly-α), and 175.2 (s, Gly α-COOH);positive APCIMS: m/z 584 [M+H]⁺.

Separation of the M13 Isomers Using Preparative HPLC.

Waters preparative HPLC systems with 2545 binary gradient module, Waters2767 sample manager, Waters 2487 autopurification flow cell, Watersfraction collector III, dual injector module, and 2489 UV/Visibledetector, were used to separate M13 isomers. A Phenomenex Gemini-NX C18column (250 mm×30.0 mm i.d., 5 μm) was used with a flow rate of 20.0mL/min. The wavelength of UV detector was set at 280 nm. The injectionvolume was 1.0 mL for each run. The mobile phase consisted of solvent A(H2O+0.1% formic acid) and solvent B (MeOH+0.1% formic acid).

M13 (5 mg/mL) was injected to the preparative column and eluted with agradient solvent system (0% B from 0 to 5 min; 0 to 50% B from 5 to 15min; 50 to 60% B from 15 to 25 min; 60 to 80% B from 25 to 45 min; then0% B from 45 to 50 min). The fractions were checked by a HPLC-APCI-MSsystem and then combined. A total of 7 runs resulted in 10 mg of M13-1(t_(R) 16.5 min) and 22 mg of M13-2 (t_(R) 16.8 min).

M13-1: white solid; 1H NMR (700 MHz, CD3OD) δ 6.77 (1H, d, J=1.8 Hz,H-2′), 6.69 (1H, d, J=8.1 Hz, H-5′), 6.61 (1H, dd, J=8.1, 1.8 Hz, H-6′),2.78 (2H, m, H-1), 2.77 (2H, m, H-2), 2.76 (1H, dd, J=17.1, 7.2 Hz,H-4a), 2.65 (1H, dd, J=17.1, 6.4 Hz, H-4b), 3.10 (1H, m, H-5), 1.49 (2H,m, H-6), 1.42 (1H, m, H-7a), 1.31 (1H, m, H-7b), 1.25 (2H, m, H-8), 1.28(2H, m, H-9), 0.88 (3H, t, J=7.1 Hz, H-10), 3.82 (3H, s, OMe-3′), 3.62(1H, t, J=6.0 Hz, HGlu-α), 2.13 (2H, m, HGlu-β), 2.55 (1H, m, HGlu-γa),2.50 (1H, m, HGlu-γb), 4.49 (1H, dd, J=8.7, 5.0 Hz, HCys-α), 3.00 (1H,dd, J=13.7, 5.0 Hz, HCys-(3a), 2.73 (1H, dd, J=13.7, 8.7 Hz, HCys-βb),and 3.74 (2H, AB, J=17.2 Hz, HGly-α); 13C NMR (175 MHz, CD3OD) δ 134.0(s, C-1′), 113.2 (d, C-2′), 148.9 (s, C-3′), 145.7 (s, C-4′), 116.2 (d,C-5′), 121.8 (d, C-6′), 30.4 (t, C-1), 46.1 (t, C-2), 211.3 (s, C═O,C-3), 49.9 (t, C-4), 42.0 (d, C-5), 36.2 (t, C-6), 27.6 (t, C-7), 32.8(t, C-8), 23.6 (t, C-9), 14.4 (q, C-10), 56.4 (q, OMe-3′), 55.6 (d,CGlu-α), 27.9 (t, CGlu-β) 33.1 (t, CGlu-γ), 174.3 (s, Glu α-COOH), 175.2(s, Glu γ-CON), 55.0 (d, CCys-α), 33.2 (t, CCys-β), 172.4 (s, Cysα-CON), 44.5 (t, CGly-α), and 175.9 (s, Gly α-COOH); positive APCIMS:m/z 584 [M+H]⁺.

M13-2: white solid; ¹H NMR (700 MHz, CD₃OD) δ 6.77 (1H, d, J=1.8 Hz,H-2′), 6.69 (1H, d, J=8.1 Hz, H-5′), 6.61 (1H, dd, J=8.1, 1.8 Hz, H-6′),2.77 (2H, m, H-1), 2.76 (2H, m, H-2), 2.70 (1H, dd, J=17.1, 7.2 Hz,H-4a), 2.64 (1H, dd, J=17.1, 6.4 Hz, H-4b), 3.10 (1H, m, H-5), 1.48 (2H,m, H-6), 1.38 (2H, m, H-7), 1.25 (2H, m, H-8), 1.28 (2H, m, H-9), 0.88(3H, t, J=7.1 Hz, H-10), 3.82 (3H, s, OMe-3′), 3.64 (1H, t, J=6.0 Hz,HGlu-α), 2.14 (2H, m, HGlu-(3), 2.56 (1H, m, HGlu-γa), 2.50 (1H, m,HGlu-γb), 4.49 (1H, dd, J=8.7, 5.0 Hz, HCys-α), 2.97 (1H, dd, J=13.7,5.0 Hz, HCys-(3a), 2.81 (1H, dd, J=13.7, 8.7 Hz, HCys-βb), and 3.74 (2H,AB, J=17.2 Hz, HGly-α); 13C NMR (175 MHz, CD3OD) δ 133.9 (s, C-1′),113.2 (d, C-2′), 148.8 (s, C-3′), 145.7 (s, C-4′), 116.2 (d, C-5′),121.8 (d, C-6′), 30.4 (t, C-1), 46.0 (t, C-2), 211.3 (s, C═O, C-3), 49.6(t, C-4), 42.4 (d, C-5), 36.3 (t, C-6), 27.5 (t, C-7), 32.8 (t, C-8),23.6 (t, C-9), 14.4 (q, C-10), 56.4 (q, OMe-3′), 55.5 (d, CGlu-α), 27.9(t, CGlu-β), 33.1 (t, CGlu-γ), 174.3 (s, Glu α-COOH), 175.3 (s, Gluγ-CON), 55.1 (d, CCys-α), 33.3 (t, CCys-β), 172.4 (s, Cys α-CON), 44.3(t, CGly-α), and 175.9 (s, Gly α-COOH); positive APCIMS: m/z 584 [M+H]⁺.

Growth Inhibition of Human Cancer and Normal Cells.

Cell growth inhibition was determined by a MTT colorimetric assay. Humancolon cancer cells HCT-116, human lung cancer cells H-1299, human colonfibroblast cells CCD-18Co, and human lung fibroblast cells IMR-90 wereplated in 96-well microtiter plates with 3000 cells/well and allowed toattach for 24 h at 37° C. The test compounds (in DMSO) were added tocell culture medium to the desired final concentrations (final DMSOconcentrations for control and treatments were 0.1%). After the cellswere cultured for 48 h, the medium was aspirated and cells were treatedwith 200 μL fresh medium containing 2.41 mmol/L MTT. After incubationfor 3 h at 37° C., the medium containing MTT was aspirated, 100 μL ofDMSO was added to solubilize the formazan precipitate, and plates wereshaken gently for an hour at room temperature. Absorbance values werederived from the plate reading at 550 nm on a Biotek microtiter platereader (Winooski, Vt.). The reading reflected the number of viable cellsand was expressed as a percentage of viable cells in the control. BothHCT-116 and H-1299 cells were cultured in McCoy's 5A medium. CCD-18Coand IMR-90 cells were cultured in Eagle's modified essential medium(EMEM). All of the above media were supplemented with 10% fetal bovineserum, 1% penicillin/streptomycin, and 1% glutamine, and the cells werekept in a 37° C. incubator with 95% humidity and 5% CO₂.

TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling)Assay.

HCT-116 and H1299 cells were seeded in 6-well plates at 1×10⁵ cells/welland incubated at 37° C. in 5% CO₂ incubator. After 24 hours, fresh mediasupplemented with DMSO (control), [6]-shogaol, M2, M6, or M13metabolites (20 μM or 40 μM) were added to the wells. After 6 or 24hours incubation at 37° C. in 5% CO₂ incubator, cells were washed andpre-treated for 15 min at room temperature with a solution of 20 μg/mlproteinase K. Cells were then washed twice with phosphate buffer salinepH 7.4 (PBS) and fixed for 10 min at room temperature using 10% neutralformaldehyde solution. After 2 washes in ddH₂O, cells were resuspendedin 100 μL ddH₂O and applied on silanized microscope slides. Slides wereincubated overnight at 37° C., and washed twice with PBS. TUNEL assaywas then carried out according to the manufacturer's protocol. Cellswere observed under 400× power using a Zeiss microscope A1 (Thornwood,N.Y.). 10 fields per slide were evaluated, and TUNEL₊ cells (with browncoloration in the nucleus) were expressed as a percentage of the totalnumber of cells contained in a field.

Statistical Analysis.

For simple comparisons between two groups, two-tailed Student's test wasused. A p-value of less than 0.05 was considered statisticallysignificant in all the tests.

Experimental Part 3

Incubation with Liver Microsomes

Experiment 1:

Human liver microsomes (HLM) (either 0.1 mg/mL or 0.5 mg/mL finalconcentration) were incubated with [6]-shogaol (50 μM) for several timepoints. The experimental incubation mixture consisted of 100 mMpotassium phosphate buffer, a prepared NADPH-regenerating system, andhuman liver microsomes. In all experiments, [6]-shogaol was dissolved indimethylsulfoxide (DMSO) with a final concentration not exceeding 1%(v/v). After 5 min preincubation in a 37° C. water bath, the reactionwas initiated by the addition of [6]-shogaol and was further incubatedat 37° C. The reaction was terminated at 0, 30, 45, and 60 minutes bythe addition of ice-cold acetonitrile (equal volume) containing 2%acetic acid. The mixture was vortexed and underwent centrifugation at13,000 g for 10 minutes. Aliquots of supernatant were stored at −20° C.until analysis. Control incubations without NADPH-regenerating system,without substrate, or without microsomes were performed to ensure thatmetabolite formation was microsome- and NADPH-dependent.

Experiment 2:

Based upon the optimized conditions in experiment 1, mouse livermicrosomes (MLM), rat liver microsomes (RLM), dog liver microsomes(DLM), monkey liver microsomes (CyLM) and human liver microsomes (HLM)(each 0.5 mg/mL, respectively), were mixed with aforementionedincubation mixture and were held in 37° C. water bath for 5 minutesbefore the reactions were initiated by the addition of [6]-shogaol (50μM). Reactions were terminated after 30 minutes incubation in a 37° C.water bath by the addition of ice-cold acetonitrile (equal volume)containing 2% acetic acid. The mixture was vortexed and underwentcentrifugation at 13,000 g for 10 minutes. Aliquots of supernatant werestored at −20° C. until analysis.

Chemical Inhibition Studies:

Inhibitors ABT (500 μM) and 18β-GA (500 μM) or DMSO controls werepre-incubated with microsomes in experimental incubation mixture at 37°C. for 20 minutes before initiating the reaction by the addition of[6]-shogaol (50 μM). Reactions were terminated 30 minutes aftersubstrate addition and incubation in a 37° C. water bath by equal volumeof ice-cold acetonitrile with 2% acetic acid. The mixture was vortexedand underwent centrifugation at 13,000 g for 10 minutes. Aliquots ofsupernatant were stored at −20° C. until analysis.

Sample Preparation for LC/MS Analysis:

To elucidate the structures of the two previously uncharacterizedmetabolites of [6]-shogaol, samples from monkey liver microsomes (CyLM)incubations were enriched by partitioning with EtOAc. Briefly, alarge-scale reaction (1.0 mL) of CyLM incubated with [6]-shogaol wasperformed in which enzyme concentration was 0.5 mg/mL and [6]-shogaolconcentration was 50 μM. All other reagent relative concentrations werekept constant. After 30 minutes incubation in a 37° C. water bath,samples were treated in an identical fashion as described above tostabilize metabolites and precipitate proteins from solution. Thesupernatant was then extracted three times with ice-cold EtOAc (5×volumes each time). The pooled supernatant was dried under reducedpressure at 30° C., and the residue was resuspended in MeOH with 0.2%acetic acid for LC/MS analysis.

Synthesis of (1E,4E)-1-(4′-hydroxy-3′-methoxyphenyl)-deca-1,4-dien-3-one(M14)

[6]-Shogaol (276 mg, 1.0 mmol) was dissolved in tetrahydrofuran (THF)and cooled down to 0° C. To this mixture, a solution of DDQ (181 mg, 0.8mmol) in THF was added. The mixture was stirred at 0° C. for 30 minutesand then warmed to room temperature (RT) for 3 hours. Then water wasadded and the resulting mixture was extracted with EtOAc (×3). Theorganic phase was washed with water (×1) and brine (×1), dried overanhydrous NaSO₄, and filtered. The filtration was evaporated and theresidue was purified by chromatography column (CC) (hexane/ethylacetate=8:1 and 6:1) to give the desired compound as a yellow oil (170mg, yield: 62%); ¹H NMR (600 MHz, CDCl₃) δ 7.59 (1H, d, J=15.9 Hz, H-1),6.82 (1H, d, J=15.9 Hz, H-2), 6.44 (1H, d, J=15.6 Hz, H-4), 6.99 (1H,dt, J=15.6, 7.0 Hz, H-5), 2.27 (2H, q, J=7.0 Hz, H-6), 1.51 (2H, m,H-7), 1.34-1.29 (4H, m, H-8 and H-9), 0.90 (3H, t, J=6.8 Hz, H-10), 7.07(1H, d, J=1.4 Hz, H-2′), 6.93 (1H, d, J=8.2 Hz, H-5′), 7.14 (1H, dd,J=8.2, 1.4 Hz, H-6′), and 3.93 (3H, s, OMe-3′); ¹³C NMR (150 MHz, CDCl₃)δ 143.3 (d, C-1), 123.3 (d, C-2), 189.3 (s, C-3, C═O), 129.1 (d, C-4),148.0 (d, C-5), 32.7 (t, C-6), 27.9 (t, C-7), 31.4 (t, C-8), 22.4 (t,C-9), 14.0 (q, C-10), 127.4 (s, C-1′), 109.7 (d, C-2′), 148.1 (s, C-3′),146.8 (s, C-4′), 114.8 (d, C-5′), 122.9 (d, C-6′), and 56.0 (q, OMe-3′);positive ESI-MS, m/z 275 [M+H]⁺.

Synthesis of (E)-1-(4′-hydroxy-3′-methoxyphenyl)-dec-1-en-3-one (M15)

The general procedure disclosed herein was followed by using M11 (45 mg,0.16 mmol), prepared previously as disclosed, and DDQ (29 mg, 0.13 mmol)in THF (6 mL). The mixture was stirred at RT for 2 hours. The resultingresidue was purified by preparative TLC (hexane/ethyl acetate=5:1) togive the desired compound as a yellow oil (34 mg, yield: 77%); ¹H NMR(600 MHz, CDCl₃) δ 7.47 (1H, d, J=16.1 Hz, H-1), 6.59 (1H, d, J=16.1 Hz,H-2), 2.63 (2H, t, J=7.5 Hz, H-4), 1.66 (2H, m, H-5), 1.33-1.25 (8H, m,ranged from H-6 to H-9), 0.87 (3H, t, J=6.7 Hz, H-10), 7.08 (1H, d,J=1.6 Hz, H-2′), 6.91 (1H, d, J=8.2 Hz, H-5′), 7.09 (1H, dd, J=8.2, 1.6Hz, H-6′), and 3.92 (3H, s, OMe-3′); ¹³C NMR (150 MHz, CDCl₃) δ 142.6(d, C-1), 124.2 (d, C-2), 200.8 (s, C-3, C═O), 40.7 (t, C-4), 24.6 (t,C-5), 29.3 (t, C-6), 29.1 (t, C-7), 31.7 (t, C-8), 22.6 (t, C-9), 14.1(q, C-10), 127.1 (s, C-1′), 109.4 (d, C-2′), 148.1 (s, C-3′), 146.8 (s,C-4′), 114.8 (d, C-5′), 123.4 (d, C-6′), and 56M (q, OMe-3′); positiveESI-MS, m/z 277 [M+H]⁺.

HPLC Analysis:

The mobile phases consisted of solvent A (30 mM sodium phosphate buffercontaining 1.75% acetonitrile and 0.125% tetrahydrofuran, pH 3.35) andsolvent B (15 mM sodium phosphate buffer containing 58.5% acetonitrileand 12.5% tetrahydrofuran, pH 3.45). The gradient elution had thefollowing profile: 20-62% B from 0 to 13 min; 62% B from 13 to 39 min;62-100% B from 39 to 48 min; 100% B from 48 to 53 min; and 20% B from53.1 to 58 min. The cells were then cleaned at a potential of 1000 mVfor 1 minute. The injection volume of the sample was 10 μL. The eluentwas monitored by the Coulochem electrode array system (CEAS) withpotential settings at 0, 200, 250, 300, 350, 400 and 500 mV.

LC/MS Analysis:

LC/MS analysis was carried out with a Thermo-Finnigan Spectra System,which consisted of an Accela high-speed MS pump, an Accela refrigeratedautosampler, and an LTQ Velos ion trap mass detector (Thermo Electron,San Jose, Calif., USA) incorporated with heated electrospray ionization(H-ESI) interfaces. A Gemini C18 column (50×2.0 mm i.d., 3 μm;Phenomenex, Torrance, Calif., USA) was used for separation at a flowrate of 0.2 mL/min. The column was eluted with 100% solvent A (5%aqueous methanol with 0.2% acetic acid) for 3 minutes, followed bylinear increases in B (95% aqueous methanol with 0.2% acetic acid) to40% from 3 to 15 minutes, to 85% from 15 to 45 minutes, to 100% from 45to 50 minutes, and then with 100% B from 50 to 55 minutes. The columnwas then re-equilibrated with 100% A for 5 minutes. The LC eluent wasintroduced into the H-ESI interface. The positive ion polarity mode wasset for the H-ESI source with the voltage on the H-ESI interfacemaintained at approximately 4.5 kV. Nitrogen gas was used as the sheathgas and auxiliary gas. Optimized source parameters, including ESIcapillary temperature (300° C.), capillary voltage (50 V), ion sprayvoltage (3.6 kV), sheath gas flow rate (30 units), auxiliary gas flowrate (5 units), and tube lens (120 V), were tuned using authentic[6]-shogaol. The collision-induced dissociation (CID) was conducted withan isolation width of 2 Da and normalized collision energy of 35 for MS²and MS³. Default automated gain control target ion values were used forMS-MS³ analyses. The mass range was measured from 50 to 1000 m/z. Dataacquisition was performed with Xcalibur 2.0 version (Thermo Electron,San Jose, Calif., USA).

Kinetic Study:

To estimate kinetic parameters of metabolism of [6]-shogoal to majorproduct M6 in liver microsomes from human and other species, theincubation conditions were optimized to ensure that formation rate of M6was in the linear range in relation to incubation time and proteinconcentration. [6]-Shogaol (7.81, 15.63, 31.25, 62.5, 125, 250, 500,1000, and 2000 μM) was incubated with liver microsomes from mouse (MLM),rat (RLM), dog (DLM), monkey (CyLM), and human (HLM) (0.1 mg/mL each,respectively) for 30 minutes. All incubations were performed intriplicate. The apparent K_(m) and V_(max) values were calculated fromanalysis of experimental data according to the Michaelis-Mentenequation, and the results were graphically represented (for HLM) by anEadie-Hofstee plot. Kinetic constants were reported as the value +/−S.E.of the parameter estimate.

Growth Inhibitory Effects of M14 and M15 Against Human Colon and LungCancer Cells:

Cell growth inhibition was determined by a MTT colorimetric assay. Humancolon cancer cells HCT-116 and human lung cancer cells H-1299 wereplated in 96-well microtiter plates with 3000 cells/well and allowed toattach for 24 hours at 37° C. The test compounds (in DMSO) were added tocell culture medium to the desired final concentrations (final DMSOconcentrations for control and treatments were 0.1%). After the cellswere cultured for 24 hours, the medium was aspirated and cells weretreated with 200 μL fresh medium containing 2.41 mmol/L MTT. Afterincubation for 3 hours at 37° C., the medium containing MTT wasaspirated, 100 μL of DMSO was added to solubilize the formazanprecipitate, and plates were shaken gently for an hour at roomtemperature. Absorbance values were derived from the plate reading at550 nm on a Biotek microtiter plate reader (Winooski, Vt.). The readingreflected the number of viable cells and was expressed as a percentageof viable cells in the control. Both HCT-116 and H-1299 cells werecultured in McCoy's 5A medium. All of the above media were supplementedwith 10% fetal bovine serum, 1% penicillin/streptomycin, and 1%glutamine, and the cells were kept in a 37° C. incubator with 95%humidity and 5% CO₂.

Measurement of Induction of Apoptosis in Human Cancer Cells by M14, M15,and [6]-Shogaol:

Human colon cancer cells HCT-116 and human lung cancer cells H-1299 wereplated in 96-well plates at a density of 5000 cells/well and allowed toattach overnight at 37° C. M14, M15, or [6]-shogaol in DMSO, or DMSOcontrol, diluted in media, were added to cells and incubated for anadditional 24 hours at 37° C. After 24 hours, media containing compoundwas removed and cells were lysed in their respective wells with reagentsfrom a Cell Death Detection ELISA^(PLUS) kit from Roche Applied Science(Mannheim, Germany). Samples were harvested after cell lysates were spundown at 300 g for 10 minutes. To streptavidin coated microplates, 20 μLsamples were added, and mixed with 80 μL Immunoreagent, which consistedof anti-histone-biotin, and anti-DNA-POD, and incubated with gentleshaking for 2 hours at room temperature. After incubation, Immunoreagentwas removed and samples were washed 3 times with 250 μL incubationbuffer. Substrate solution, ABTS, was added to each well and color wasdeveloped for 15 minutes before stopping the reaction with ABTS stopsolution and reading absorbance on a microplate reader at 430 nm.Experiments were performed in triplicate and the average is given incomparison to DMSO control with standard deviation.

Experimental Part 4

A549 Culture and Reagents.

A549 cells were cultured in FK12 media (Corning, Corning, N.Y.)supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin(Gemini Bio-Products, West Sacramento, Calif.). Protease and phosphataseinhibitor mix was from Thermo Scientific (Waltham, Mass.). Antibodiesfor Western blotting were from Cell Signaling (Danvers, Mass.). Proteinconcentrations were determined from cell lysates using a Pierce BCA kit(Thermo Fisher Scientific, Rockford, Ill.). BrdU(5-bromo-2-deoxyuridine) was from Sigma-Aldrich (St Louis, Mo.). Apoptagplus Peroxydase In Situ Apoptosis Detection Kit was from Millipore(Billerica, Mass.), and the BrdU Immunohistochemistry Kit was fromChemicon International (Temecula, Calif.).

Metabolism of 6S and M2 in A549 and IMR90 Cells

A549 or IMR90 cells (1.0×10⁶) were plated in 6-well culture plates andallowed to attach for 24 hours at 37° C. in 5% CO₂ incubator. 6S or M2(in DMSO) was then added to culture media to reach a final concentrationof 10 or 20 μM, respectively. At different time points (0, 30 minutes,1, 2, 4, 8, and 24 hours), 190 μL samples of supernatant were taken andtransferred to vials containing 10 μL. of 0.2% acetic acid to stabilize6S, M2, and their respective metabolites. To extract compounds from theculture media, an equal volume of acetonitrile was added to thesupernatant samples and these mixtures were centrifuged. The supernatantwas harvested and the samples were analyzed by HPLC-ECD.

Determination of Cell Viability

A549 cell viability was determined by a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)colorimetric assay. A549 cells (6000 cells/well) were plated in 96-wellmicrotiter plates and allowed to attach for 24 hours at 37° C. and 5%CO₂. 6S or M2 (in DMSO) were added to cell culture medium to desiredfinal concentrations (0-80 μM; final DMSO concentrations for control andtreatments were 0.1%). After the cells were cultured for 24 hours, themedium was aspirated and the cells were treated with 2.41 mM MTT infresh media. After incubation for 3 hours at 37° C., the mediumcontaining MTT was removed, 100 μL of DMSO was added to the wells, andthe plates were shaken gently for an hour at room temperature.Absorbance values were derived from the plate reading at 550 nm on aBiotek Synergy 2 plate reader (Winooski, Vt.). The experiment wasrepeated independently to confirm the results.

Determination of Apoptosis

The Cell Death Detection ELISA (Enzyme-linked immunoabsorbant assay)Plus kit from Roche (Mannheim, Germany) was used. A549 cells (10000cells/well) were plated in 96-well microtiter plates and allowed toattach for 24 hours at 37° C. and 5% CO₂. 6S or M2 (in DMSO) was addedto cell culture medium to desired final concentrations (10 and 20 μM;final DMSO concentrations for control and treatments were 0.1%). After24 hours, the microplate was centrifuged for 10 minutes at 1200 rpm, andthe supernatant was removed. 200 μl of the lysis buffer was added ineach well and incubated for 30 minutes at room temperature. The platewas then centrifuged for 10 minutes at 1200 rpm and 20 μl of thesupernatant was transferred to streptavidin-coated micro-wells. ELISAassay was performed according to manufacturer's instruction. Absorbancein each well was measured at 405 nm in absorbance units (AU), and theenrichment factor (EF) in small nucleosomes was calculated with theformula EF=AU treated/AU DMSO. The experiment was repeated independentlyto confirm the results.

Intracellular Glutathione (GSH) Measurement

The total GSH content was measured using a HT Glutathione Assay kit(Trevigen, Gaithersburg, Md.). Briefly A549 cells were plated in 60×15mm culture plates and were allowed to attach overnight at 37° C. Cellswere treated with 10 μM M2 and incubated for 0, 2, 4, 8, or 24 hours.Cells were harvested and proteins were precipitated with 5% (w/v)metaphosphoric acid. Samples were then processed following themanufacturer's instructions. The measurement of the absorbance of5-thio-2-nitrobenzoic acid (TNB) at 405 nm was used to quantifyglutathione levels in each sample, which was then compared to thestandard curve and corrected for protein concentration. The experimentwas repeated independently to confirm the results.

For measurement of oxidized glutathione (Glutathione Disulfide or GSSG),samples and GSSG standards were treated with 2M 4-vinylpyridine (1 μL/50μL sample) at room temperature for one hour. 4-Vinylpyridine (SigmaAldrich, St. Louis, Mo.) blocks free thiols present in the reaction,consequentially blocking the formation of new GSSG by GSH. The 2Msolution was freshly prepared by diluting 4-vinylpyridine in ethanol ina ratio of approximately 1:3.6. After incubation, samples were processedusing the Trevigen kit's protocol and absorbance was measured at 405 nmas described herein.

The quantity of reduced cellular glutathione (or GSH) is obtained bysubtracting the oxidized samples values from the total glutathionevalues or: GSH_((reduced))=GSH_((total))−GSSG_((oxidized)). Ratios ofreduced to oxidized glutathione are shown to further represent cellularredox status after treatment with 6S or M2. The experiment was repeatedindependently to confirm the results.

Western Blotting

Cell extracts were prepared by incubating cells for 5 minutes on icewith RIPA (Radio-Immunoprecipitation Assay) buffer (Thermo FisherScientific, Rockford, Ill.) supplemented with a protease and phosphataseinhibitor mix. Cell lysate was then centrifuged at 13,000 rpm at 4° C.for 20 minutes, and supernatant was harvested for Western blot analysis.Briefly, 30-60 μg of protein extract were separated on a 10-16%polyacrylamide gel and transferred on PVDF (polyvinylidene difluoride)membrane (Biorad, Hercules, Calif.). Membrane was blocked using a 1%casein solution in TBS-Tween 20. Primary rabbit antibodies were dilutedin blocking solution and incubated with the membrane overnight at 4° C.After washing the membrane with 3 changes of TBS-T, secondary HorseRadish Peroxydase (HRP)-conjugated anti-rabbit antibody was diluted1:3000 in blocking solution and incubated with PVDF membrane for 1 hourat room temperature. Signal was then revealed using FEMTOchemoluminescent substrate (Thermo Scientific, Waltham, Mass.) and byexposing the membrane to photosensitive photographic films for varioustimes. Films were developed using a SRX-101A Konica Minolta developer(Tokyo, Japan). The experiment was repeated independently twice toconfirm the results. Fold-induction of proteins calculated bynormalizing the band of interest to the loading control (β-actin), andthis adjusted intensity what compared to the control (DMSO) sample.

GSH Rescue Assay

A549 cells were plated on 60 mm culture dishes, at 0.5×10⁶ density.After 24 hours, DMSO, 6S or M2 (10, 20, 40, 80, 120 μM) were added tothe cells and incubated with or without the addition of 5 mM GSH in theculture media. After 24 hours, toxicity was assessed using the MTT assayand using the method described above. The experiment was repeatedindependently to confirm the results.

Animal Experiments

Nu/J nude mice were obtained from Jackson Laboratories (Bar Harbor,Me.). Animals were randomized into 4 groups. A549 cells (5×10⁶ cells)were implanted in both flanks of 8-weeks old Nu/J mice. One week afterimplantation, animals were given 100 μl of the following treatmentsthrough oral gavage 5 times/week: DMSO 0.25 ml/kg (control;n=4); 6S 10mg/kg (n=4); 6S 30 mg/kg (n=4) or M2 30 mg/kg (n=5). Compounds werediluted in a solution of 5% DMSO in corn oil. Animal body weight andtumor volume were recorded for the duration of the experiment. Tumorvolume was calculated by measuring the length and width of the tumorsusing a digital caliper and using the formula (Length×Width²)/2. Onehour before sacrifice, mice were given one last treatment dose as wellas one intra-peritoneal injection of BrdU (7.5 mg/kg in 100 μl PBS).After 7 weeks, tumor tissues were harvested and weighed. A portion ofthe tumors was snap frozen in liquid nitrogen and another portion wasplaced in a histology cassette and immersed in formalin solution.

Immunohistochemistry

Paraffin-fixed tissues were sent to Precision Histology Lab (OklahomaCity, Okla.) for embedding in paraffin blocks. Then paraffin blocks wereprocessed into 3-4 μm sections that were then put on microscope slides.Sections were then deparaffinized by using a succession of 3 baths ofxylene (5 minutes each), 2 baths of absolute ethanol (5 minutes each),95% ethanol for 3 minutes, 70% ethanol for 3 minutes, and rinsed in PBS.Immunostaining with TUNEL (terminal deoxynucleotidyl transferase dUTPnick end labeling) and BrdU staining kits was performed followingmanufacturer's recommendation. For staining quantification, sequentialhigh-power field pictures of tumors were taken (10 pictures per tumor)using an A1 Zeiss microscope (Oberkochen, Germany). Images wereprocessed using the Image J software, which was used to count positive,brown-colored cells in each field. Average number per tumor wascalculated by averaging the number obtained for each field, and theaverage number of positive cells per group was obtained by averaging thevalues of each tumor belonging to the experimental group.

Statistical Analysis

Statistics were calculated using either a two-tailed Student t-test, orANOVA followed by Bonferroni's post-test. Results were consideredsignificant when p<0.05.

Experimental Part 5

Chemical Synthesis of M2′ and M2″

The experimental procedure to synthesize M2′ and M2″ was similar to thatof M2, as described herein. In brief, a catalyst amount of NaHCO₃ (1.3mg, 0.015 mmol) was added to a mixture of 8S (91.2 mg, 0.3 mmol) andcysteine (54 mg, 0.45 mmol) in methanol/water (6 mL, 1:1, v/v). Themixture was stirred at room temperature for 24 hours, adjusted to pH 6with a diluted acetic acid solution (0.1 M). The mixture was thenpurified by preparative HPLC to give a white solid M2′ (70 mg, yield55%). In the preparation of M2″, 10S (100 mg, 0.3 mmol) was used inplace of 8S in the above reaction: M2″ (73 mg, yield 54%).

Purification of M2′ or M2″ Using Preparative HPLC

Waters preparative HPLC systems with 2545 binary gradient module, Waters2767 sample manager, Waters 2487 auto-purification flow cell, Watersfraction collector III, dual injector module, and 2489 UV/Visibledetector, were used to separate M2′ or M2″ from the reaction mixture. APhenomenex Gemini-NX C₁₈ column (250 mm×30.0 mm i.d., 5 μm) was usedwith a flow rate of 20.0 mL/min. The wavelength of UV detector was setat 280 nm. The injection volume was 1.0 mL for each run. The mobilephase consisted of solvent A (H₂O+0.1% formic acid) and solvent B(MeOH+0.1% formic acid). Reaction mixture of M2′ was injected to thepreparative column and eluted with a gradient solvent system (75 to 87%B from 0 to 12 min; to 75% B from 12 to 12.5 min; then with 75% B from12.5 to 15 min). A total of 6 runs resulted in 70 mg of M2′ (t_(R) 9.45min). Similarly, the reaction mixture of M2″ was injected to thepreparative column and eluted with a gradient solvent system (85 to 100%B from 0 to 15 min; then with 100% B from 15 to 16 min; to 85% B from 16to 16.5 min; then with 85% B from 16.5 to 20 min). A total of 7 runsresulted in 73 mg of M2″ (t_(R) 8.13 min).

Metabolism of 8S, 10S, M2′ and M2″ in Human Colon Cancer Cells

Cells (1.0×10⁶) were plated in six-well culture plates and were allowedto attach for 24 hours at 37° C. in 5% CO₂ incubator. 8S or 10S in DMSO,or the corresponding cysteine-conjugated metabolites M2′ and M2″ werediluted in McCoy's 5A medium (containing 10% fetal bovine serum, 1%penicillin/streptomycin, and 1% glutamine) to reach a finalconcentration of 10 μM and were incubated with different colon cancercell lines (HCT-116 or HT-29). At different time points (0, 2, 4, 8, 24,and 48 hours), 190 μL samples of supernatant were taken and transferredto vials containing 10 μL of 2% acetic acid to stabilize these compoundsand their respective metabolites. An equal volume of acetonitrile wasadded to the samples before centrifugation. The supernatant washarvested and the samples were then analyzed by HPLC-ECD.

Evaluation of Toxicity in Human Colon Cancer and Normal Colon Cells

Cell viability was determined by an MTT colorimetric assay. Briefly,human colon fibroblast cells CCD-18Co or human colon cancer cellsHCT-116 or HT-29, were plated in 96-well microtiter plates with 3000cells/well and allowed to attach for 24 hours at 37° C. and 5% CO₂. Thetest compounds (in DMSO) were added to cell culture medium to desiredfinal concentrations (final DMSO concentrations for control andtreatments were 0.1%). After the cells were cultured for 24 hours, themedium was aspirated and cells were treated with 200 μL fresh mediumcontaining 2.41 mmol/L MTT. After incubation for 3 hours at 37° C., themedium containing MTT was aspirated, 100 μL of DMSO was added tosolubilize the formazan precipitate, and the plates were shaken gentlyfor an hour at room temperature. Absorbance values were derived from theplate reading at 550 nm on a Biotek (Winooski, Vt.) microtiter platereader. The reading reflected the number of viable cells and wasexpressed as a percentage of viable cells in the control. CCD-18Co cellswere grown in EMEM. Both HCT-116 and HT-29 cells were cultured inMcCoy's 5A medium. All of the above media were supplemented with 10%fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine, andthe cells were kept in a 37° C. incubator with 95% humidity and 5% CO₂.

Apoptosis Analysis

Apoptosis was determined by FACS analysis of propidium iodide(PI)-stained cells. In brief, cells were trypsinized, washed with coldphosphate-buffered saline (PBS), fixed in ice-cold 70% ethanol for 1hour, and then resuspended in 2 mL PBS supplemented with 10 μL RNase(100 mg/ml) and incubated at 37° C. for 30 min. After incubation, DNAwas stained with 1 mg/mL PI in PBS. Cell staining was analyzed using aCell Lab Quanta™ SC flow cytometer (Beckman Coulter, Danvers, Mass.) anddata were processed using FCS Express software (DeNovo Software, LosAngeles, Calif.). The percentage of apoptotic cells in each sample wasdetermined based on the sub G₀ peaks detected in monoparametrichistograms.

Measurement of Reactive Oxygen Species

The assay employed the cell-permeable fluorogenic probe2′,7′-dichlorodihydrofluorescin diacetate [DCFH-DA] (Sigma Aldrich, St.Louis, Mo.) to measure the relative changes in O⁻ ₂ and H₂O₂ levels inHCT-116 or HT-29 cells after treatment with 5, 10, and 20 μM 6S or M2(or DMSO) over 0, 2, 4, 8, and 24 hours. In brief, DCFH-DA is diffusedinto cells and is deacetylated by cellular esterases to non-fluorescent2′,7′-dichlorodihydrofluorescin (DCFH), which is rapidly oxidized tohighly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF) byintracellular hydrogen peroxide, or other low molecular weightperoxides. Measured fluorescence intensity is thus proportional to theamount of such peroxides in the cell at a given time. Human colon cancercells HCT-116 or HT-29 were seeded in 96-well black-sided,clear-bottomed culture plates, with 5000 cells/well and were allowed toadhere for 24 hours in a 37° C. incubator with 5% CO₂. Media wasaspirated and 5, 10, or 20 μM M2, 6S, or DMSO diluted in media wereadded to designated wells, which were run in triplicate. After desiredincubation times of 0, 2, 4, 8, or 24 hours, media and test compoundswere aspirated. Cells were washed three times with 200 μL PBS beforeaddition of 100 μL 1 mM DCF-DA. The fluorogenic probe permeated cellmembranes and was processed to DCF for one hour at 37° C. Afterincubation, plates were immediately placed in a Biotek microplate readerto measure fluorescence at wavelengths of 485 (excitation) and 528(emission). Raw values were normalized to DMSO control for each timepoint and are presented as fold induction versus 0 hour time point(n=3).

Western Blot Analysis

Cell lysates were prepared in ice-cold RIPA lysis buffer [25 mM Tris-HCl(pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS,Thermo Fisher Scientific] supplemented with a protease inhibitorcocktail (AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A inDMSO with EDTA, Thermo Fisher Scientific). Protein content was measuredby a Pierce BCA Assay Kit (Thermo Fisher Scientific). Protein contentsof cell lysates (30 μg protein/lane) were resolved by SDS-PAGE. Proteinswere then electro-transferred onto PVDF membranes and blots were blockedfor one hour at room temperature in 1×TBS with 1% Casein (Bio-RadLaboratories, Berkeley, Calif.). Blots were then incubated overnight at4° C. with the desired primary antibody diluted in TBS with 0.5%Tween-20. Blots were then washed with TBS-Tween 20 and probed for 1 hourwith the appropriate secondary antibody (1:1000). Protein bands werevisualized with chemiluminescence using West Femto maximum detectionsubstrate (Thermo Fisher Scientific). To confirm equal protein loadingin each lane, immunoblots were stripped and re-probed for β-actin.Protein fold-induction was calculated by normalizing the intensity ofthe band of interest to β-actin first, and then to DMSO control lanes.

Colony Formation Assay

Human colon cancer cells HCT-116 or HT-29 (1,000 cells per well) wereseeded in 6-well culture plates for 24 hours and then incubated with M2(0, 1, 5, 10, 20, or 40 μM) in DMSO in a 37° C. incubator with 5% CO2.After 2 weeks, colonies were washed with phosphate-buffered saline(PBS), then stained with a mixture of 6.0% glutaradehyde and 0.5%crystal violet for 30 min at room temperature, rinsed in water,air-dried and then photographed.

Statistical Analysis

Student's t-test or two-way analysis of variance (ANOVA) with theBonferroni post-test were used to determine the statistical significanceof data, which was performed on GraphPad Prism version 5.00 for Windows(GraphPad Software, San Diego, Calif.).

Results

Metabolism of [6]-Shogaol in Mice.

In the study, HPLCECD and LC/ESI-MS were used to analyze the majormetabolites of [6]-shogaol in the collected samples. Compared with thesamples collected from control mice, 12 major metabolites (M1□M12) wereobserved in fecal samples collected from [6]-shogaol-treated mice. Thesemetabolites were numbered according to their chromatographic retentiontimes. Incubation of the fecal sample extracts with glucuronidase andsulfatase did not change the peak areas of all the metabolites,suggesting these compounds do not exist in glucuronidated and/orsulfated forms, whereas in urinary and plasma samples, most of themetabolites were not detectable without incubation with glucuronidaseand sulfatase. These results suggest that the metabolites in the urineand plasma were in the glucuronidated and/or sulfated forms. Afterhydrolysis, the plasma samples and urine samples showed similarmetabolic profiles to those of fecal samples, as confirmed by LC/MSanalysis. Seven major metabolites (M6-M12) were purified from fecalsamples collected from mice treated with 200 mg/kg [6]-shogaol usingoral gavage. Their structures were elucidated on the basis of analysisof their ¹H, ¹³O, and ²D NMR spectra. For the metabolites not purifiedfrom mouse fecal samples (M1-M5), structures were determined usingLC/ESI tandem mass spectrometry (MS/MS) by analyzing the MS' (n=1-3)spectra as well as by comparison with authentic standards. Among all themetabolites, M1 through M5, M10, and M12 are the thiol conjugates of[6]-shogaol and its metabolite M6.

Structure Elucidation of Non-Thiol-Conjugated Metabolites (M6 Through M9and M11).

Metabolite M6.

M6 had the molecular formula C₁₇H₂₆O₃ according to ESI-MS at m/z 261[M+H−H₂O]⁺ and its ¹H and ¹³C NMR data. The molecular weight of M6 was 2mass units higher than that of [6]-shogaol. In addition to thedistinguishable resonance for a methoxyl group (□H 3.84, 3H, s), the ¹HNMR spectrum of M6 (Table 1) also indicated the presence of a1,3,4-tri-substituted phenyl group [□H 6.77 (1H, br s); 6.70 (1H, br d,J □ 7.8 Hz); and 6.63 (I H, br d, J=7.8 Hz)], and a double bond [□H 5.47(1H, dd, J=15.4, 7.1 Hz) and 5.64 (I H, dt, J=15.4, 7.1 Hz)], and amethyl group (□H 0.92, 3H, t, J=7.2 Hz). Its ¹³C NMR spectrum displayed17 carbon resonances, which were classified by heteronuclear singlequantum correlation experiments as two methyls, six methylenes, sixmethines, and three quaternary carbons. The aforementioned NMR dataimplied the structure of M6 was closely related to that of [6]-shogaol.The only difference was that C-3 of M6 was assigned as an oxymethine (□H3.99, 1H, m; □C 71.6) instead of the expected ketone carbonyl in[6]-shogaol (□C 201.5). This was confirmed by the heteronuclearmultiple-bond correlations (HMBC) of H-3/C-1, H-3/C-2, H-4/C-3, andH-5/C-3. Therefore, the structure of M6 was determined as shown in FIG.1.

Metabolite M7.

M7 showed the molecular formula C₁₈H₂₈O₄ on the basis of ESI-MS at m/z291 [M+H−H₂O]⁺ and its ¹H and ¹³C NMR data. The molecular weight of M7was 32 mass units higher than that of [6]-shogaol. Compared with[6]-shogaol, the NMR spectra of M7 gave the appearance of an oxygenatedmethine (□H 3.67, 1H, m; □C 77.1), a methylene (□H 2.66, dd, J=16.2, 7.8Hz; 2.42, dd, J=16.2, 4.8 Hz), and a methoxyl (□H 3.30, 3 H, s; □C 56.9)groups instead of the expected double bond at C-4 and C-5 of[6]-shogaol, which indicated that the □,□-unsaturated keto-structure of[6]-shogaol was reduced to a saturated ketone. This was furtherconfirmed by the observation of the HMBCs between □H 2.66 and 2.42 (themethylene group) and C-2 (□C 45.8), C-3 (□C 209.0), and C-6 (□C 33.8),indicating that the methylene group was located at position C-4. Theoxygenated methine was located at C-5 by the observation of the HMBCsbetween H-4 and □C 77.1. The HMBC between □H 3.30 (the methoxyl group)and C-5 (□C 77.1) suggested that the methoxyl group was directly linkedwith C-5. Thus, M7 was identified as shown in FIG. 1.

Metabolite M8.

M8 showed the molecular formula C₁₆H₂₄O₃ on the basis of ESI-MS at m/z265 [M+H]⁺ and its ¹H and ¹³C NMR data. Compared with [6]-shogaol, theNMR spectra of M8 showed the disappearance of the double bond at C-4 andC-5, as well as the methoxyl group at 0-3′ (Table 1), which indicated M8was 3′,4′-dihydroxyphenyl-decan-3-one (FIG. 1).

Metabolite M9.

M9 was obtained as a white amorphous powder. M9 was shown to have themolecular formula C₁₇H₂₈O₃ on the basis of ESI-MS at m/z 263 [M+H−H₂O]⁺and its ¹H and ¹³C NMR data. The molecular weight of M9 was 2 mass unitshigher than that of M6, indicating that M9 was the double-bond-reducedproduct of M6. This was further confirmed by the observation of theappearance of two methene groups (□H 1.48, 2 H and □C 37.6 and □H 1.30,2 H and □C 29.4) and the disappearance of the double-bond signals in M9(Table 1). Therefore, the structure of M9 was determined as1-(4″-hydroxy-3″-methoxyphenyl)-decan-3-ol (FIG. 1).

Metabolite M11.

M11 was obtained as a white amorphous powder. It showed a protonatedmolecular ion at m/z 279 [M+H]⁺, which was 2 mass units higher than thatof [6]-shogaol. The ¹H and ¹³C NMR data of M11 were very similar tothose of [6]-shogaol, and the major difference was that M11 had twomethene groups (□H 2.38, 2 H; □H 1.56, 2 H) (Table 1) instead of thedouble bond in [6]-shogaol, clearly indicating that M11 was thedouble-bond-reduced metabolite of [6]-shogaol. This was furtherconfirmed by the key correlations observed in the HMBC spectrum.Therefore, M11 was identified as1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-one, also known as [6]-paradol,which is one of the components reportedly found in ginger.

Structure Elucidation of Thiol-Conjugated Metabolites (M1□M5, M10, andM12).

Metabolite M5.

The mass spectrum of metabolite M5 exhibited [M+H]⁺ ions at m/z 440 inthe positive mode, which was 163 mass units higher than that of[6]-shogaol, indicating that M5 was the N-acetylcysteine conjugated[6]-shogaol (molecular weight of N-acetylcysteine is m/z 163). The MS2spectrum of M5 showed a major product ion at m/z 277. The MS3 spectrumof this product ion had the same fragment ions as those of the authentic[6]-shogaol, indicating that M5 was an N-acetylcysteine conjugate of[6]-shogaol. To further elucidate the structure of M5, it wassynthesized by reacting N-acetylcysteine with 6-shogaol. The structureof the synthesized N-acetylcysteine conjugate(5-N-acetylcysteinyl-[6]-shogaol) was determined using its ¹H, ¹³C, and2D NMR data. The ¹H and ¹³C NMR spectra showed very similar patterns tothose of [6]-shogaol (Table 2). Compared with the ¹H NMR spectrum of[6]-shogaol, the major differences were the appearance of a methine (□H2.74, m, 2H) and a methylene (□H 3.14, m, 1H) group in5-N-acetylcysteinyl-[6]-shogaol in lieu of the expected double bond of[6]-shogaol, as well as four additional proton signals for aN-acetylcysteine group (□H 3.00 dd and 2.92 dd, H-1″; □H 4.58 dd, H-2″;and □H 2.01 s, H-5″). The major differences between the ¹³C spectra of5-N-acetylcysteinyl-[6]-shogaol and [6]-shogaol were the presence ofcarbons observed at □C 46.1 (C-4) and 42.6 (C-5) instead of the doublebond of [6]-shogaol, as well as the presence of five additional carbonsat □C 30.4 (C-1″), 54.6 (C-2″), 173.1 (C-3″ and C-4″), and 22.6 (C-5″)for a N-acetylcysteine group (Table 2). The HMBC spectrum of5-N-acetylcysteinyl-[6]-shogaol had cross-peaks between H-1″ (□H 3.00and 2.92) and C-5 (□C 42.6), indicating that N-acetylcysteine wasconjugated at C-5 of [6]-shogaol. All of these spectral featuressupported the structure of 5-N-acetylcysteinyl-[6]-shogaol. M5 hadalmost the same retention time as well as the same molecular mass andfragment ion mass spectra as those of the synthetic5-Nacetylcysteinyl-[6]-shogaol. Therefore, M5 was identified as5-Nacetylcysteinyl[6]-shogaol.

Metabolite M2.

M2 had a molecular weight of 397 as determined by the mass ion at m/z398 [M+H]⁺, which was 121 mass units higher than that of [6]-shogaol and42 mass units lower than that of M5, indicating that M2 was the cysteineconjugated metabolite of [6]-shogaol. The major product ion of M2 showeda fragment ion at m/z 277 and the tandem mass of this product ion wasalmost identical to the tandem mass of authentic [6]-shogaol. All ofthese spectral features were consistent with M2 as5-cysteinyl-[6]-shogaol.

Metabolites M1, M3, and M4.

M1 exhibited [M+H]⁺ ions at m/z 400 in the ESI-positive mode, which was2 mass units higher than that of M2 and 121 mass units higher than thatof M6, indicating that M1 was the cysteine conjugated metabolite of M6.This was confirmed by the observation of m/z 261 [M−121−H₂O+H]⁺ as oneof the major product ions in the MS2 spectrum of M1. The tandem massspectrum of this product ion (MS3: 261/400) was almost identical to theMS2 spectrum of authentic M6. Thus, M1 was identified as 5-cysteinyl-M6.

M4 showed [M+H]⁺ ions at m/z 442 in the positive mode, which was 42 massunits higher than that of M1, 163 mass units higher than that of M6, and2 mass units higher than that of M5, suggesting that M4 was theN-acetylcysteine conjugated metabolite of M6. Its MS2 spectrum also hadproduct ion m/z 261 [M−121−H₂O+H]⁺, and the tandem mass spectrum of thisproduct ion (MS3: 261/442) was almost identical to the MS2 spectrum ofauthentic M6. All of these spectral features were consistent with M4 as5-N-acetylcysteinyl-M6.

M3 had a molecular weight of 456 on the basis of the observation of the[M+H]⁺ ions at m/z 457 in the positive mode, which was 178 mass unitshigher than that of M6 and 57 mass units higher than that of M1. Thiscorresponded with the predicted molecular weight of thecysteinylglycine-conjugated metabolite of M6. Similar to that of M1 andM4, the MS3 spectrum of the product ion m/z 261 of M3 was almostidentical to the MS2 spectrum of authentic M6. Thus, M3 was identifiedas the cysteinylglycine conjugate of M6 (FIG. 1).

Metabolite M10.

M10 had the molecular formula C₁₈H₂₈O₃S on the basis of ESI-MS at m/z325 [M+H]⁺ and its ¹H and ¹³C NMR data, which was 48 mass units higherthan that of [6]-shogaol. Compared with the NMR spectra of [6]-shogaol,the NMR spectra of M10 showed signals for a methine (□H 3.04, m, 1H; □C41.6), a methane (□H 2.59, dd and 2.69 dd, 2H; □C 48.5), and a methyl(□H 2.04, 3H, s; □C 13.3) group (Table 2) instead of the expected doublebond of [6]-shogaol. The chemical shifts of the methine and methanegroups were similar to those of positions 4 and 5 of M5, and thechemical shift of the methyl group was similar to that reported for themethylthiol group. In addition, a cross-peak in the HMBC spectrum wasobserved between □H 2.04 (the methyl group) and □C 41.6 (the methanegroup). Thus, M10 was identified as the methylthiol-conjugated[6]-shogaol.

Metabolite M12.

The positive ion ESI-MS of M12 displayed a molecular ion peak at m/z 327[M+H]⁺, supporting a molecular formula of C₁₈H₃₀O₃S. The molecularweight of M12 was 2 mass units higher than that of M10, which wassimilar to the difference between M6 and [6]-shogaol. Compared with theNMR spectra of M10, M12 showed the signal of an oxygenated methine (□H4.00, 1H, m; □C 68.9) in lieu of the expected ketone group of M10, whichindicated the ketone group at C-3 of M10 was reduced to a hydroxyl groupof M12, which was further confirmed by the HMBCs of OH-3/C-3 andOH-3/C-4. Therefore, M12 was identified as the methylthiol-conjugated M6(FIG. 1).

Metabolism of [6]-Shogaol in Cancer Cells.

After incubation of [6]-shogaol with four different cancer cell lines(HCT-116, HT-29, H-1299, and CL-13), the culture media were collected atdifferent time points and analyzed by HPLC-ECD. The results indicatethat [6]-shogaol was extensively metabolized in all four cancer celllines. After 24-h incubation, four major metabolites appeared in HCT-116human colon cancer cells. Three of them were identified as M6, M9, andM11 by comparing their retention times and tandem mass fragments withthose of purified authentic standards. The fourth metabolite (M13) was anewly revealed compound at the retention time of 14.50 min. The massspectrum of metabolite M13 exhibited [M+H]⁺ ions at m/z 584 in thepositive mode, which was 307 mass units higher than that of [6]-shogaol,indicating that M13 was the GSH-conjugated [6]-shogaol (molecular weightof GSH is m/z 307). Its MS² spectrum showed product ions of m/z 277(−307 Da, neutral loss of GSH), m/z 455 (−129 Da, neutral loss ofpyroglutamic acid), m/z 437 (−147 Da, dehydrolyzation of m/z 455), andm/z 509 (−75 Da, neutral loss of glycine). The MS³ spectrum of itsproduct ion m/z 277 was almost identical to the MS² spectrum ofauthentic [6]-shogaol. All of the above evidence indicates M13 is theglutathiol conjugate of [6]-shogaol. Both M9 and M11 were detected asthe major metabolites of [6]-shogaol in HT-29 human colon cancer cells,H-1299 human lung cancer cells, and CL-13 mouse lung cancer cells. At 24h, [6]-shogaol was almost completely converted to M9 and M11 in H-1299cells and to M9 in CL-13 cells.

M9 and M11 Inhibit the Growth of Human Cancer Cells.

Two cancer cell lines, HCT-116 and H-1299, were treated with[6]-shogaol, M9, or M11, with concentrations ranging from 0 to 80 □M. InHCT-116 cells, [6]-shogaol exhibited the strongest inhibitory activitywith an IC₅₀ of 18.7 □M. The major metabolites M9 and M11 had decreasingpotencies of 82.2 and 84.0 □M, respectively. In H-1299 cells, the IC₅₀values for [6]-shogaol, M9, and M11 were 16.9, 77.7, and 66.5 □M,respectively. These data demonstrate that [6]-shogaol has the greatestinhibitory activity against cancer cell lines but still shows someefficacy after metabolic biotransformation.

M9 and M11 Trigger Apoptosis in Human Cancer Cells.

Apoptosis, or programmed cell death, is a major mechanism of regulationallowing cells to undergo cell death upon activation of specificexternal and/or internal pathways. The role of [6]-shogaol, M9, and M11on the induction of apoptosis in human cancer cells was investigatedusing the TUNEL assay, which detects breaks of DNA strands in early andlate apoptotic cells. In HCT-116 and H-1299 cells, exposure to 10 □M[6]-shogaol (FIGS. 2C and 2D) yielded 10.3 and 5.2% of apoptotic cells,respectively, whereas 20 □M [6]-shogaol yielded 31.2 and 31.6%. Exposureto 40 □M metabolite M9 for 24 h led to the observation of 9.6 and 7.4%of apoptotic cells, respectively (16.9 and 15.4% for the 80 □M dose,respectively). Exposure to 40 □M metabolite M11 led to the observationof 12.9 and 8.3% of apoptotic cells in HTC-116 and H-1299 cancer cells(21.1 and 19.4% for the 80 □M dose), respectively). All of these resultsare different from the DMSO control and show that M9 and M11 arebioactive compounds and can specifically trigger apoptosis in both humancolon and lung cancer cells, but that the compounds are not as efficientas [6]-shogaol.

Chemical Synthesis of the Metabolites of [6]-Shogaol.

As generally described herein, twelve metabolites (M1, M2, and M4-M13)were synthesized successfully from [6]-shogaol using simple and easilyaccessible semisynthetic approaches in the current study (Schemes 1 and2). In brief, reaction of [6]-shogaol with L-cysteine (Cys),N-acetyl-L-cysteine (N-Cys) or L-glutathione (GSH), generatedthiol-conjugates M2, M5, or M13, respectively. Subsequently, reductionof thioconjugates M2 or M5 by NaBH₄ led to hydroxylated conjugates M1 orM4, respectively. Selective reduction of [6]-shogaol by a combination ofNaBH₄ and CeCl₃.7H₂O resulted in M6 (Scheme 2). Hydrogenation of[6]-shogaol on Pd/C gave M11, followed by treatment with NaBH₄ toproduce M9 (Scheme 2). In addition, demethylation of M11 using BBr₃ gaveM8 (Scheme 2). Michael reaction of [6]-shogaol with NaOMe or NaSMeproduced the methoxy adduct M7 or the methylthio adduct M10,respectively. The methylthio adduct M10 was treated with NaBH₄ to giveM12. Since both the reduction of ketone and the Michael reactions usedin this study are non-stereoselective, metabolites M1, M2, M4-M7, M9,M10, M12, and M13 are synthesized as mixtures of diasteromers.

The structures of M5-M12 were fully characterized using their 1-D and2-D NMR and mass spectral data as disclosed herein. The structures ofthese synthetic compounds were confirmed by comparison of their ¹H and¹³C NMR spectra with those of authentic standards obtained from mousefecal samples. Structures of the remaining synthetic metabolites (M1,M2, M4, and M13), deduced by multi-stage mass spectrometry techniques,were further confirmed by their 1-D and 2-D NMR spectra data.

Metabolite M2:

M2 showed the molecular formula C₂₀H₃₁NO₅S on the basis of positiveAPCI-MS at m/z 398 [M+H]+ and its ¹H and ¹³C NMR data. The molecularweight of M2 was 42 mass units less than that of N-acetylcysteineconjugated [6]-shogaol (M5) indicating M2 was the cysteine conjugated[6]-shogaol. This was in agreement with the fact that M2 was made by[6]-shogaol and L-cysteine. This was also supported by the observationof the absence of an acetyl group in the ¹H and ¹³C NMR spectra of M2.The linkage of an L-cysteinyl moiety to the [6]-shogaol residue at C-5was established by HMBC cross-peaks between HCys-β (δH 3.18 and 2.84)and C-5 (δC 42.3). Therefore, M2 was confirmed to be5-cysteinyl-[6]-shogaol.

Metabolite M1:

M1 had the molecular formula of C₂₀H₃₃NO₅S on the basis of positiveAPCI-MS at m/z 400 [M+H]+ and its ¹H and ¹³C NMR data. The molecularweight of M1 was 2 mass units higher than that of M2, matching with thefact that M1 was a ketone-reduced product of M2, and also supported bythe appearance of oxygenated methines (two sets of protons for thediasteromers at δH 3.66 and δH 3.90; and δC 69.3) in its ¹H and ¹³C NMRspectra. Key HMBC correlations between H-3 (δH 3.66 and δH 3.90) to C-1(δC 32.5) and C-5 (δC 43.8), as well as H-1 (δH 2.68 and 2.58) to C-3(δC 69.3) in M1, established a hydroxyl group at C-3 on the alkyl sidechain of M1. HMBC cross-peaks between HCys-β (δH 3.15 and 2.85) to C-5(δC 43.8), and H-5 (δH 2.94) to CCys-β (δC 32.8) provided the linkage ofthe cysteinyl moiety and C-5 position of M1 through a thioether bond.Thus, M1 was confirmed to be 5-cysteinyl-M6.

Metabolite M4:

M4 showed the molecular formula C₂₂H₃₅NO₆S on the basis of positiveAPCI-MS at m/z 442 [M+H]+ and its ¹H and ¹³C NMR data. The molecularweight of M4 was 2 mass units higher than that of M5(5-N-acetylcysteinyl-[6]-shogaol), complying with the fact that M4 was aketone-reduced product of M5. This was further supported by theappearance of oxygenated methines (two sets of protons for thediasteromers at δH 3.70 and δH 3.88; and δC 69.3) in its 1H and 13C NMRspectra, the disappearance of the expected ketone carbonyl group in[6]-shogaol, and the key HMBC correlations observed at H-3 (δH 3.70 andδH 3.88) to C-1 (δC 32.5) and C-5 (δC 43.8), as well as H-1 (δH 2.67 and2.58) to C-3 (δC 69.3) in its HMBC spectrum. The acetyl group was shownattached to α-NH₂ of the cysteinyl moiety by HMBC correlation detectedat HCys-α (δH 4.54) to CH3CO (δC 174.0). In addition, HMBC cross-peaksbetween HCys-β (δH 3.00 and 2.80) and C-5 (δC 43.8) provided the linkageof an acetylcysteinyl moiety and the C-5 position of M4. M4, thereof,was confirmed to be 5-N-acetylcysteinyl-M6.

Metabolite M13:

M13, having the molecular formula C₂₇H₄₁N₃O₉S on the basis of positiveAPCI-MS at m/z 584 [M+H]⁺ and its NMR data, was made by [6]-shogaol andreduced L-glutathione (GSH). ¹H-¹H COSY cross-peaks found atHGlu-α/HGlu-β/HGlu-γ, in combination with key HMBC correlations betweenHGlu-α (δH 3.65) to Glu α-COOH (δC 174.0) as well as HGlu-γ (δH 2.55 and2.51) to Glu γ-CON (δC 175.2), recognized the structure of a glutamylresidue (Glu). The structure of the cysteinyl residue (Cys) wasestablished by ¹H-¹H COSY cross-peaks at HCys-α/HCys-β in combinationwith HMBC correlation between HCys-(3 (two sets of protons at OH3.05-2.95 and 2.84-2.80) to Cys α-CON (δC 175.2). Subsequently, theconnection of the glutamyl residue with the cysteinyl moiety wasestablished between Glu γ-COOH and Cys α-NH² through an amide bond, byHMBC correlations found at HCys-α (δH 4.50) to Glu γ-CON (δC 175.2). Theattachment of a glycinyl moiety to the cysteinyl residue was foundbetween Cys α-COOH and Gly α-NH₂, by HMBC correlations observed atHGly-α (δH 3.80) to Cys α-CON (δC 175.2). Thus, the GSH residue wasundoubtedly identified as γ-glutamyl-cysteinylglycine. Consequently,linkage of the GSH moiety to the [6]-shogaol residue was established atC-5 through a thioether bond by HMBC correlations found at HCys-β (twosets of protons at δH 3.05-2.95 and 2.84-2.80) to C-5 (δC 42.2).Therefore, M13 was confirmed to be 5-glutathiol-[6]-shogaol.

Separation of M13 isomers on preparative HPLC resulted in twodiastereoisomers, M13-1 and M13-2, which had very similar ¹H and ¹³C NMRspectra. The major differences were the 1H signals for HCys-βb (δH 2.73in M13-1 vs. 2.81 in M13-2) and H-4a (δH 2.76 in M13-1 vs. 2.70 inM13-2) and the ¹³C signals for C-4 (δC 49.9 in M13-1 vs. 49.6 in M13-2)and C-5 (δC 42.0 in M13-1 vs. 42.4 in M13-2). In the NOESY spectra,correlations between HCys-βb (δH 2.73) and H-5 (δH 3.10) were observedfor M13-1 and between HCys-βa (δH 2.97) and H-5 (δH 3.10) for M13-2,suggesting that H-5 in M13-1 had the same configuration as that ofHCys-βb and H-5 in M13-2 had the same configuration as that of HCys-pa.It is known that HCys-α in GSH residue has the R configuration. Thecoupling constant (J=5.0 Hz) of HCys-βa (δH 2.97) with HCys-α (δH 4.49)is much smaller than that (J=8.7 Hz) of HCys-βb (δH 2.81) with HCys-α,suggesting that HCys-βa (δH 2.97) has the same R configuration as thatof HCys-α and HCys-βb has the S configuration. Therefore, theconfigurations of H-5 in M13-1 and M13-2 were tentatively assigned as Sand R, respectively.

Growth Inhibitory Effects Against Human Cancer and Normal Cells.

Two human cancer cell lines, HCT-116 and H-1299, were treated with[6]-shogaol or its synthetic metabolites M1, M2, and M4-M13, withconcentrations ranging from 0 to 80 μM. Cell viability assays utilizingMTT resulted in eight active metabolites, M2, M5, M6, M8-M11, and M13,against colon cancer cells HCT-116, with IC50 values of 24.43, 54.26,68.77, 58.76, 82.22, 78.16, 83.97, and 45.47 μM, respectively (FIG. 3),and eight active metabolites, M2, M5, M6, and M9-M13, in lung cancercells H-1299, with IC50 values of 25.82, 72.62, 61.28, 82.50, 60.63,66.50, 69.91, and 47.77 μM, respectively (FIG. 4). Among them, M2, thecysteine conjugated metabolite of [6]-shogaol, was found to be mostpotent toward both HCT-116 and H-1299 cells with IC50 values of 24.43and 25.82 μM, respectively, which was comparable to the parent[6]-shogaol, with an IC₅₀ of 18.20 μM in HCT-116 cells and an IC₅₀ of17.90 μM in H-1299 cells. The second most active metabolite was5-glutathionyl-[6]-shogaol (M13), with IC₅₀ values of 45.47 and 47.77 μMin HCT-116 and H-1299 cells, respectively.5-N-acetylcysteinyl-[6]-shogaol (M5) also exhibited bioactivity withIC₅₀ values of 54.26 □M in HCT-116 cells and 72.62 □M in H-1299 cells.This metabolite, however, displayed decreased activity when compared tothat of 5-cysteinyl-[6]-shogaol (M2), suggesting the acetylation onα-NH₂ of the cysteinyl moiety diminishes the activity of M2. Moreover,the reduction of a ketone group on the alkyl side chain resulted inlittle to no activity against cancer cells HCT-116 and H-1299, asobserved from M1 and M4 versus M2 and M5, as well as M6, M9, and M11versus [6]-shogaol, suggesting that the reductive biotransformation of[6]-shogaol and its metabolites was primarily inactivating.

Evaluation of cytotoxicity in human normal fibroblast colon cellsCCD-18Co and human normal lung cells IMR-90 showed that all syntheticmetabolites (M1, M2, and M4-M13) were less toxic than parent[6]-shogaol, and most of them had little to no inhibitory effect,indicating a detoxifying metabolic biotransformation of [6]-shogaol. Themetabolite M2, having the greatest potency against both HCT-116 andH-1299 cancer cells, showed almost no toxicity towards normal coloncells CCD-18Co and normal lung cells IMR-90 with IC₅₀ values of 99.18and 98.30 μM, respectively, compared to those of parent [6]-shogaol withan IC₅₀ of 43.91 μM toward normal colon cell line CCD-18Co and an IC₅₀of 36.65 μM toward normal lung cell line IMR-90. Moreover, M13, withIC₅₀ values of 75.50 □M and 57.54 □M against cells CCD-18Co and IMR-90,respectively, displayed lower toxicity compared to parent [6]-shogaol.

To investigate the influence of stereochemistry on activity, metaboliteM13, as a mixture of diastereomers, was separated by reverse phaseprep-HPLC into two individual isomers, M13-1 and M13-2. Cancer cellsHCT-116 and H-1299 were treated with M13 or its constituentstereoisomers (M13-1 and M13-2) individually, with concentrationsranging from 0 to 80 μM. Both isomers had similar activity, which wasslightly less than M13; M13-2 was slightly more effective than M13-1,with an IC₅₀ value of 54.90 □M in HCT-116 cells and 63.77 □M in H-1299cells, versus 71.20 □M and 74.39 □M in the same respective cell lines(FIG. 5).

Reconstituted M13 by combining M13-1 and M13-2 in an approximate molarratio of 1:2, which is similar to the ratio in original M13, displayedthe equivalent activity, with IC₅₀ values of 46.46 □M in HCT-116 celland 41.98 □M in H-1299 cell, compared to the original M13 with IC₅₀values of 45.47 □l M in HCT-116 cells and 47.77 □l M in H-1299 cells,indicating that the observed growth inhibitory effect of M13 is notattributable to one isomer or the other.

M2 and M13 Induce Apoptosis in Human Cancer Cells.

Cells lines H-1299 and HCT-116 were incubated with M2, M13, M6 and[6]-shogaol at various concentrations to determine the active range ofthese compounds versus DMSO. (FIG. 6). After 24 hours of incubation,metabolite M6 did not display any apoptotic effect in HCT-116 and H-1299cell lines. Apoptosis was observed for M2 and M13 in both cell lines,except for M2 in H-1299 cells for the 20 μM concentration. The inductionof apoptosis by [6]-shogaol was superior to that of both M2 and M13 atthe same concentration (20 μM). In H-1299 cells, an equivalent apoptoticeffect to [6]-shogaol could be obtained if the concentration of M2 andM13 was twice (40 μM) that of [6]-shogaol. Similar results were obtainedin HCT-116 cells, except that the M2 apoptotic induction was higher at40 μM. In all cell lines, an increase in the concentration of[6]-shogaol, M2, M13, M13-1, M13-2 or M6 yielded a correspondingincrease in apoptotic level in cancer cells (FIG. 6).

Each of [6]-shogaol, M2 and M13 were incubated in HCT-116 cells for only6 hours (FIG. 6). After 6 hours of incubation with [6]-shogaol ormetabolites M2 or M13, higher levels of apoptosis for all 3 compoundswere detected compared to DMSO in HCT-116 cells. Interestingly there wasno significant difference between the apoptotic effect of [6]-shogaoland the effects of M2 at 20 and 40 μM and M13 at 40 μM. M13 was morepotent than [6]-shogaol at 20 μM. Exposure of HCT-116 cells to M13isomers M13-1 and M13-2 also showed a higher level of apoptosis, but theisomers' apoptotic effect was inferior compared to [6]-shogaol for bothconcentrations used. These results show that apoptosis is triggered by[6]-shogaol metabolites M2, M13, M13-1 and M13-2, and is one mechanismat least partially responsible for the cell death observed previously.

Incubation Conditions of [6]-Shogaol with Human Liver Microsomes

Transformation of [6]-shogaol in liver microsomes occurred more rapidlyin the condition containing 0.5 mg/mL human liver microsomes than in thecondition with 0.1 mg/mL microsomes. The profiles were comparable, withno noticeable difference besides the aforementioned metabolic velocity.After examining the profiles from different conditions, the incubationparameters were optimized for metabolite identification and interspeciescomparison with hepatic microsomes at 0.5 mg/mL along withNADPH-regenerating system for 30 minutes.

Structure Elucidation of [6]-Shogaol Metabolites

Five major product peaks were observed in LC chromatograms after[6]-shogaol (50 μM) was incubated for 30 minutes with the hepaticmicrosomes from mouse, rat, dog, monkey, and humans. All of the peakswere identified by comparing their MS² spectra with those of authenticstandards.

Metabolites M6, M9 and M11:

Metabolites at the retention time of 29.3, 33.9, and 41.8 min wereidentified as 1-(4′-hydroxy-3′-methoxyphenyl)-4-decen-3-ol (M6),1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-ol (M9), and1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-one (M11), respectively, bycomparing their mass spectra with those of authentic standards that werepurified from mouse feces as described before.

Metabolite M14

To identify the other two unknown peaks (M14 and M15), the sampleobtained from CyLM was run on LC/MS, which indicated that M14 had amolecular weight of m/z 274 as determined by the mass ions at m/z 275[M+H]⁺, 297 [M+Na]⁺, and 571 [2M+Na]⁺, which was 2 mass units less thanthat of [6]-shogaol. This metabolite showed m/z 177 but not 137 (production of [6]-shogaol) as a major product ion, leading to a conclusion thatthe compound was a 1,2-dehydrogenated product of [6]-shogaol. Toconfirm, the compound was independently synthesized. The syntheticcompound had a molecular formula C₁₇H₂₂O₃ on the basis of positiveESI-MS at m/z 275 [M+H]⁺ and its ¹H and ¹³C NMR data. The molecularweight of this compound (m/z: 274) was 2 mass units less than that of[6]-shogaol (m/z: 276) indicating it was dehydrogenated [6]-shogaol.This was also supported by the observation of the absence of twomethenes and the presence of two more olefinic methines (δ_(H) 7.59 andδ_(H) 6.82; and δ_(C) 143.1 and δ_(C) 123.3) in its ¹H and ¹³C NMRspectra. Additionally, geometry of the double bond between C-1 and C-2was determined as E-configuration by the coupling constant J_(1,2)=15.9Hz. Therefore, this compound was confirmed as(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)deca-1,4-dien-3-one. M14 had almostthe same retention time as well as the same mass fragment as those ofthe synthetic(1E,4E)-1-(4′-hydroxy-3′-methoxyphenyl)-deca-1,4-dien-3-one and wasassigned as that compound (FIG. 1).

Metabolite M15

In the same way, another peak showed a molecular weight of m/z 276 asdetermined by the mass ions at m/z 277 [M+H]⁺, 299 [M+Na]⁺, and 575[2M+Na]⁺. M15 had the same molecular weight as that of [6]-shogaol,suggesting that it was a double bond transferred metabolite of[6]-shogaol. To confirm, this compound was synthesized using M11 asdescribed in the experimental section. The synthetic compound had amolecular formula C₁₇H₂₄O₃ on the basis of positive ESI-MS and its ¹Hand ¹³C NMR data. The molecular weight of this compound was 2 mass unitsless than that of M11, coinciding with the fact that it was adehydrogenated product of M11, and also supported by the appearance oftwo olefinic methines (δ_(H) 7.47 and δ_(H) 6.59; and δ_(C) 142.6 andδ_(C) 124.2) in its ¹H and ¹³C NMR spectra. Additionally, theE-configuration between C-1 and C-2 was determined by the couplingconstant J_(1,2)=16.1 Hz. Therefore, this compound was confirmed to be(E)-1-(4′-hydroxy-3′-methoxyphenyl)-dec-1-en-3-one. M15 had almost thesame retention time as well as the same mass fragment as those of thesynthetic (E)-1-(4′-hydroxy-3′-methoxyphenyl)-dec-1-en-3-one, and wasassigned as that compound (FIG. 1).

Metabolism of [6]-Shogaol by Liver Microsomes from Different Species

The metabolic profiles of [6]-shogaol in liver microsomes from mouse(MLM), rat (RLM), dog (DLM), and monkey (CyLM) were similar to those inhuman (HLM) as the five major metabolites, M6, M9, M11, M14, and M15were detected in all five species. Monkey liver microsomes gave thehighest abundance of oxidized metabolites after 30 minutes incubation,in stark contrast to human liver microsomes, which gave the least. Theseresults suggest stronger interspecies differentiation in the enzymesresponsible for [6]-shogaol oxidative metabolism than its reductivemetabolism, which is seemingly conserved.

Chemical Inhibition Studies

To determine the inclusion of CYP-450 enzymes in [6]-shogaol metabolism,ABT, a broad-specificity P450 inactivator, was applied to incubations ofliver microsomes with [6]-shogaol. In humans the oxidative metabolismwas abrogated yet reductive metabolism still occurred. This result wassimilar in mouse, rat, dog, and monkey. Without being bound by theory,CYP-450 enzymes were implicated in oxidative metabolism of [6]-shogaol.As the broad-specificity P450 suicide substrate ABT did not inhibitreductive metabolism in all species, it was clear that non-P450 enzymeswere implicated. Application of 18β-glycyrrhetinic acid, a knownaldo-keto reductase inhibitor to the reaction mixture of HLM decimatedthe reductive metabolism of [6]-shogaol.

Kinetics

Over the concentration range tested, metabolism of [6]-shogaol to M6 inliver microsomes from humans and four animal species obeyedMichaelis-Menten kinetics, as evidenced by the Eadie-Hofstee plot (FIG.7). The kinetic parameters, including K_(m), V_(max), and the intrinsicclearance (V_(max)/K_(m)), were determined and are listed in Table 3. Inhuman liver microsomes, the K_(m) value for M6 formation was 45.6 μM andthe V_(max) was 10.82+/−0.47 nmoles per minute per milligram microsomalprotein. In liver microsomes from four experimental animals, the K_(m)values for M6 formation ranged from 75.7 μM (mouse) to 424.2 μM(monkey), displaying vast interspecies variation. The V_(max) values forthe remaining four species varied as well, with 2.99+/−0.28 (monkey) to8.19+/−0.48 (rat) nmoles per minute per milligram enzyme, a kinetictrend that was consistent with the perceived relative formation rates ofM6 across five species in the HPLC-ECD chromatogram. Intrinsic clearancevalues varied across all five species, from 7.0 μL per minute permilligram protein (monkey) to 80.5 μL per minute per milligram protein(rat), indicating the relative affinities of M6 formation from lowest tohighest in CyLM and RLM, respectively.

TABLE 3 Kinetic parameters of M6 metabolism in liver microsomes: K_(m)values are in micromolar values. V_(max) values are nanomoles per minuteper milligram liver microsomes. The range of substrate concentrationswas 7.8 to 2000 μM. Each value is the mean +/− S.D. of three independentexperiments. Species V_(max) K_(m) V_(max)/K_(m) Human 10.82 +/− 0.47237.2 +/− 32.7  45.6 Monkey  2.99 +/− 0.28 424.2 +/− 108.3 7.0 Dog  7.33+/− 0.43 251.1 +/− 42.99 29.2 Rat  8.19 +/− 0.48 101.7 +/− 22.62 80.5Mouse  4.10 +/− 0.19  75.7 +/− 12.17 54.2Growth Inhibitory Effects Against Human Cancer and Normal Cells

Two human cancer cell lines, HCT-116 and H-1299, were treated with[6]-shogaol or its synthetic metabolites M14 and M15, withconcentrations ranging from 0 to 80 μM. Cell viability assays utilizingMTT resulted in high potency of M14 and moderate potency of M15,relative to [6]-shogaol, with IC₅₀ values of 3.22, 43.02, and 19.94 μM,respectively, against colon cancer cells HCT-116 (FIG. 8A). In lungcancer cells H-1299, a similar trend was observed with M14, M15, and[6]-shogaol, with IC₅₀ values of 3.04, 41.59, and 17.32 μM, respectively(FIG. 8B).

Induction of Apoptosis in Human Cancer Cells by M14, M15, and[6]-Shogaol

Following the implications of cytotoxicity in cancer cells induced byM14, M15, and [6]-shogaol, the pro-apoptotic properties of thesecompounds were measured (FIGS. 8C and 8D). M14 was the most potentcompound, with induction of apoptosis of about 6-fold in HCT-116 cellsand about 7-8 fold in H-1299 cells, compared to DMSO control afteradministration of 10 kM compound and 24 hours incubation. Treatment of 1μM M14 gave a 2-fold induction of apoptosis in HCT-116 cells and 1-foldinduction in H-1299 cells, compared to DMSO control. Metabolite M15 gavesimilar, if somewhat greater apoptotic effects against both cancer celllines after treatment for 24 hours, as parent compound [6]-shogaol.Administration of 10 μM M15 or [6]-shogaol was sufficient in both celllines to induce apoptosis, by 1-fold in HCT-116 and almost 2-fold inH-1299. The similarities in pro-apoptotic potencies between [6]-shogaoland M15 was not entirely expected, as [6]-shogaol was more than twice astoxic in the previous MTT assays. However, as apoptosis is a specificform of cell death, the results are not unreasonable.

Xenograft Study

8 week-old nude mice (Jackson Laboratory, Maine) were randomized into 5groups based on treatment (DMSO: n=4; 6S 10 mg/kg: n=4 (“6S 10”); 6S 30mg/kg: n=4 (“6S 30”); M2 30 mg/kg: n=5 (“M2 30”); M14 10 mg/kg: n 4(“M14 10”)). Animals were implanted with 5×10⁶ A549 lung cancer cells(adenocarcinomic human alveolar basal epithelial cells) on each flank.The mice had access to food and water ad libitum. One week postimplantation, the animals started receiving their respecting treatmentsthrough oral gavage (1000 in corn oil and 5% DMSO), 5×/week. After 7weeks of treatment, the tumor tissues were harvested. Reduction in tumorsize was observed between the control group and the 6S 30, M2 30 and M1410 groups (FIG. 9). Additionally, the M2 group showed a 12.9% lowertumor burden than the equivalent 6S group compared to DMSO, equivalentto a 21.8% decrease. Based on the results of this xenograft study, 6Sinhibits lung tumor growth in a dose-dependent manner.

6S and M2 are Similarly Metabolized by IMR90 and A549 Cells

6S is metabolized by IMR90 or A549 cells, with an initial conversioninto mostly metabolites identified as M2, M13 and M11, while in latertime points most of 6S has been metabolized into M9. The structures ofall metabolites were confirmed using LC/MS analysis. As reported inHCT-116 and H-1299 cells, M2 metabolism in IMR90 or A549 cells wascharacterized by an initial conversion of this cysteine-conjugatedmetabolite back into 6S, which is then metabolized in a similar pattern.Thus, normal lung IMR90 and lung cancer A549 cells quickly metabolize 6Sand M2 in a similar pattern.

6S and M2 Influence GSH Levels in A549 Cells

FIGS. 10A and 10B show that GSH levels are significantly depleted asearly as 2 hours after exposure to 10 μM of 6S or M2; this depletioncontinues after 4 hours. After 8 hours, GSH levels were stillsignificantly lower in 6S treated cells, and significantly higher in M2treated cells. After 24 hours, GSH levels were significantly increasedafter exposure to both compounds compared to baseline. While changes ofGSH levels is not as large as those of 6S in the case of M2, it isnonetheless significant for all tested time points.

The results of the GSH/GSSG assay are presented in FIGS. 10C (6S) and10D (M2) and show that after 2 hours the GSH/GSSG ratio is significantlylower for 6S-treated (10 μM) cells but not for M2-treated (10 μM) cells.After 4 hours of exposure to 6S or M2, the GSH/GSSG ratio issignificantly lower and after 24 hours of exposure it is significantlyhigher. Without being bound by theory, these results show that both 6Sand M2 can deplete GSH levels and induce oxidative stress in A549 cellsin a similar fashion.

M2 Toxicity can Selectively Discriminate Between Normal and Cancer CellsCompared to 6S

The bioactivity of 6S and M2 in A549 cells as well as in IMR90 normalhuman lung cells were compared in an MTT assay (FIG. 11A). When treatedwith increased concentration of 6S or M2, an increase in toxicity inA549 cells with IC₅₀'s of 25.2 and 30.4 μM, respectively, was observed.In normal IMR90 cells, the IC₅₀ was 36.6 and 98.3 μM for 6S and M2,respectively. Thus, in normal cells the IC₅₀ value was 45.6% higher for6S and 223.2% higher for M2 when compared to A549 cells, suggesting that6S and M2 exert similar toxicity towards A549 cells. However, M2toxicity is greatly diminished against normal cells compared to that of6S.

Cell/Compound IC50's (μM) A549 6S 25.17 IMR90 6S 36.65 A549 M2 30.41IMR90 M2 98.36S and M2 Activate Apoptosis and p53 Pathways

An ELISA assay quantified the release of cytoplasmic histone-associatedDNA fragments in A549 cells exposed to 6S and M2 for 24 hours (FIG. 11).FIG. 11B shows that after 24 hours these apoptotic markers were higher(enrichment factor of 2.2) for cells treated with 20 μM of 6S. For M2(FIG. 11C) an increase in apoptotic markers (about 3-fold enrichment)for the 20 μM concentration was observed.

Western blot analyses were conducted on extracts of A549 cells treatedwith 20 or 40 μM of 6S or M2 for 2 or 24 hours. For both concentrationsof 6S and M2, the pro-apoptotic markers cytochrome C, cleaved caspase 3and 9 were elevated after 2 hours. Only cleaved caspases 3 and 9 levelsremained elevated after 24 hours, especially at the 40 μM concentration.A small increase of caspases 3 and 9 after 2 hours of exposure was alsonoted, and these levels were lower after 24 hours. Markers of themitochondrial apoptotic pathway Bax, Bak and Bcl-2 were all slightlyelevated after 2 hours of exposure to the test compound but their levelswere close to that of DMSO-treated cells after 24 hours.

An investigation of the p53 pathway, which is responsive to oxidativestress and capable of triggering apoptosis, showed an increase in p53levels after 2 and 24 hours for both 6S and M2, correlated to anincrease of one of its downstream effectors PUMA (p53 upregulatedmodulator of apoptosis), which was most evident after 24 hours. Thus,both 6S and M2 activate the p53 and apoptosis pathway.

Excess GSH can Rescue A549 Cells from 6S and M2 Toxicity and Suppressp53 Activation

Excess GSH in the culture media rescued A549 cells from both 6S and M2toxicity, with modified IC₅₀'s over 80 μM (FIG. 12A). Western blotanalysis showed that in the presence of excess GSH, there was no changein p53 expression in the 24 hours extracts of cells treated with 40 μM6S or M2. Changes in GSH levels induced by both 6S and M2 in A549 cellsare necessary to induce toxicity and the p53 pathway.

The Transcription-Independent Mitochondrial p53 Pathway is Involved in6S and M2-Induced Toxicity and Apoptosis

After determining the p53 involvement in A549 apoptosis and the weakmodulation of the Bcl-2 family members (such as Bax), the p53-specificinhibitor pifithrin μ (pft), which specifically blocks the directinteraction and mitochondrial relocation of p53 with members of theBcl-2 family, was investigated. Treatment of A549 cells with pft inaddition to 6S was effective in reducing the toxic effect of both 6S(FIG. 12B) and M2 (FIG. 12C). When treated with 20 μM of 6S or M2 for 24hours, the percentage of viable cells was close to 100% for 6S and 84.2%for M2. Without pft to block p53 interaction with Bcl-2 family members,the percentage of viable cells was around 72% in both cases. This effectwas also observed at a higher dose of compound (40 μM). In the case of6S, the percentage of viable cells was higher (20.6% of viable cellswithout pft and 41% of viable cells with pft). For 40 μM M2 the effectwas similar, with 30.6% of viable cells without pft and 42.4% of viablecells with pft. Interference with p53 signaling can at least partiallyrescue cells from 6S and M2-induced toxicity.

Since pft directly interferes with p53 signaling towards themitochondria, the effect of pft on apoptosis induction in A549 cells wasstudied. For this experiment, a 20 μM dose was used. Treatment with pftreduced the enrichment factor in small nucleosomes by a factor of 1 forboth 6S and M2 (FIG. 12D): in the case of 6S the presence of pftreturned the enrichment factor to baseline (about 1), while in the caseof M2 the enrichment factor is down to 2 with pft from 3 without pft.

6S and M2 induce cell apoptosis through the modulation of GSH levels andthe activation of the transcription-independent mitochondrial p53pathway.

6S and M2 can Reduce A549 Cells Tumor Burden in Nu/J Mice

The effect of 6S and M2 on the development of A549 tumors in a mousexenograft model was investigated. Exposure to a daily oral gavage ofanimals for up to 7 weeks did not induce any significant changes in bodyweight between groups. Tumor volume in the control group grewexponentially, tumors from the groups receiving 6S 10 mg/kg, 6S 30 mg/kgor M2 30 mg/kg grew slower, with tumors from the M2 group beingdifferent by week 7 (FIG. 13A). After 7 weeks, tumor weight was lower inboth 6S 30 mg/kg (minus 40.8% compared to DMSO-treated control) and M230 mg/kg (minus 53.7% compared to DMSO-treated control). Tumor weightwas also lower in the 6S 10 mg/kg group (minus 25.6%), albeit notsignificantly (FIG. 13B). Taken altogether, these results show that 6Sand M2 exposure does not induce toxicity in animal. Both treatments weresufficient to lower the tumor burden of A549 engrafted cells at a 30mg/kg body weight.

6S and M2 Induce Apoptosis and Reduce Cell Proliferation in A549Xenografts

TUNEL staining of tumor tissues showed a marked increase of apoptoticbodies in the animals treated with 6S 10 mg/kg body weight (27.8 TUNEL₊cells/field) compared to control (about 15.5 TUNEL₊ cells/field). Thistrend became significant in the tumors from animals treated with 6S 30mg/kg, with an average of 32.6 TUNEL₊ cells/field. In the case of theanimals treated with M2 30 mg/kg, the same trend (28.6 TUNEL₊cells/field) was observed (p=0.0669). BrdU staining of tumor tissuesshowed a reduction of cell proliferation in the animals treated with 6S30 mg/kg body weight (3.4 BrdU₊ cells/field) compared to control (about6.3 BrdU₊ cells/field). A marked reduction of cell proliferation in boththe 6S 10 mg/kg group (4.7 BrdU₊ cells/field) was also observed(p=0.0678 by unpaired t-test compared to control). While there was alsoa slight decrease in the M2 30 mg/kg group (5.7 BrdU₊ cells/field), itwas also very close to significance (p=0.0558 by unpaired t-testcompared to control). Thus, generally the reduction in tumor burden invivo can be correlated to the induction of apoptosis for 6S and M2 andin the case of 6S it can be associated to other molecular mechanisms,such as cell proliferation.

Cysteine Conjugation of a Ginger Extract with High Shogaols″.

Concentration of Shogaols in Ginger Extract

One gram ginger extract with high shogaols typically contains 259 mg 6S(0.94 mmol), 35.5 mg 8S (0.12 mmol), and 79 mg 10S (0.24 mmol). (Totalshogaols: 1.3 mmol).

Procedure for Michael Addition Reaction of Ginger Extract and Cysteine.

A catalyst amount of NaHCO₃ (12 mg, 0.14 mmol) was added to a mixture ofginger extract with high shogaols (1 g including 1.3 mmol shogaols) andL-cysteine (173 mg, 1.1 eq) in methanol/water (20 mL; 10:10, v/v). Themixture was stirred at room temperature (rt) for 24 h. Then 1 mL HOAcsolution (1 M) was added to the reaction mixture. The reaction mixturewas then dried out by rotary evaporator. The residue was chromatographedon a silica gel column eluted with a mixture of CHCl₃/MeOH (3:1 and 100%MeOH). The MeOH eluted fraction give cysteine-conjugated shogaols, M2,M2′ and M2″.

Synthesis and Structure Elucidation of M2′ and M2″

A similar experimental procedure was used for the syntheses of M2′ andM2″ as the previous protocol used for M2. M2′ showed the molecularformula C₂₂H₃₅NO₅S on the basis of positive ESI-MS at m/z 426 [M+H]⁺ andits ¹H and ¹³C NMR data. The molecular weight of M2′ was 121 mass unitsmore than that of 8S (M. W.: 304), indicating M2′ was thecysteine-conjugated 8S, which is an expected result from the reactionbetween 8S and L-cysteine. This was also supported by the observation ofthe absence of a double bond in the ¹H and ¹³C NMR spectra of M2′.Therefore, M2′ was confirmed to be 5-cysteinyl-8S. In the same way, M2″was identified as 5-cysteinyl-10S, based on its positive ESI-MS at m/z454 [M+H]⁺ and its ¹H and ¹³C NMR data (Table 4).

TABLE 4 δ_(H) (600 MHz) and δ_(C) (150 MHz) NMR spectra data of M2′ andM2″ (CD₃OD, δ in ppm and J in Hz). M2′ M2″ δ_(H) multi (J) δ_(C) δ_(H)multi (J) δ_(C)  1 2.81 m 31.6 2.81 m 31.8  2 2.81 m 48.1 2.81 m 48.1  3210.4 210.4  4 2.88 m 53.8 2.89 m 53.8 2.81 m 2.81 m  5 3.11 m 40.9 3.11m 40.9  6 1.56 m 34.9 1.56 m 34.9  7 1.42, m 27.4 1.42, m  8 1.31, m31.8 1.31, m 31.8  9 1.31, m 29.2^(a) 1.31, m 29.3^(a) 10 1.31, m28.9^(a) 1.31, m 29.3^(a) 11 1.31, m 28.9^(a) 1.31, m 29.2^(a) 12 1.31,m 26.4^(a) 1.31, m 29.1^(a) 13 0.92 t (6.9) 22.3 1.31, m 28.9^(a) 1413.0 1.31, m 26.4^(a) 15 1.31, m 22.3 16 0.92 t (6.9) 13.0  1′ 132.4132.4  2′ 6.80 d (2.0) 111.7 6.80 d (2.0) 111.8  3′ 147.8 147.7  4′144.4 144.4  5′ 6.71 d (7.98) 115.0 6.71 d (7.98) 115.0  6′ 6.65 dd120.4 6.65 dd 120.5 (7.98,2.0) (7.98, 2.0)  1″ a: 3.21 m 32.0 a: 3.21 m32.1 b: 2.81 m b: 2.81 m  2″ 3.66 m 54.9 3.66 m 54.9  3″ 171.1 171.2 OMe3.85 s 55.1 3.85 s 55.0 ^(a)Assignments interchangeable.M2′ and M2″ Give Similar Metabolic Profiles as Parent Compounds 8S and10S

Metabolic profiles of 8S and 10S in human colon fibroblast cellsCCD18Co, human colon cancer cells HCT-116, and HT-29 correlated to theprofiles of M2′ and M2″ in the same respective cell lines, contributingto the identification of M2′ and M2″ as carriers of their respectiveshogaols. Upon removal of the cysteine residue, which occurs after lessthan two hours of treatment, M2′ and M2″ are metabolized in an almostidentical fashion as their parent shogaols. M9 and M11 are the majormetabolites of 6S in cancer cells; M11 is the double bond reducedmetabolite of 6S; and M9 is the ketone group reduced metabolite of M11.Reduced products were also identified as the major metabolites of 8S and10S in human colon fibroblast cells and cancer cells, M9′ and M11′ for8S and M9″ and M11″ for 10S (FIG. 1). Their structures were confirmed bycomparing their MS/MS spectra with those of M9 and M11.

M2′ and M2″ Exert Similar Bioactivities as their Parent Compounds 8S and10S

The results of the MTT assay show that 8S and 10S and their respectivecysteine-conjugated metabolites M2′ and M2″ have low toxicity in normalcolon cells CCD-18Co (FIG. 14A), with IC₅₀ values of 104.66 and 135.53μM for 8S and 10S, respectively, and IC₅₀ values greater than 200 μM forM2′ and M2″. All compounds were highly potent against human colon cancercells HCT-116 (FIG. 14B) and HT-29 (FIG. 14C), with slightly differentefficacy profiles between the two types of cells. M2′ and M2″ showhigher activity against HCT-116 than their parent molecules, with IC₅₀values of 15.21 and 13.28 respectively, versus values of 22.8 and 25.09for 8S and 10S, respectively. Similar results were observed in the p53mutant HT-29 cell line, albeit slightly more resistant to treatment fromall compounds. The IC₅₀ values for 8S and 10S, 27.88 and 23.92 μM, wereabout 10 to 16% lower than those for M2′ and M2″, at 31.15 and 28.75 μM,respectively.

M2 Induces Apoptosis in Human Colon Cancer Cells HCT-116 and HT-29

To study the impact of M2 on induction of apoptosis in both HCT-116 andHT-29 colon cancer cells, the percent of apoptotic cells were quantifiedafter 24 hours treatment of increasing doses of M2. A dose-dependenteffect of M2 was observed with HCT-116 cells being notably moresensitive to M2 treatment than HT-29 cells. Treatment of 40 μM M2 gavethe greatest induction of apoptosis in HCT-116 and HT-29 cells, with26.34 or 14.38% apoptotic cells, respectively. The 10 and 20 μMtreatments also yielded twice as many apoptotic HCT-116 cells comparedto HT-29 cells, respectively.

Induction of apoptosis was further confirmed by Western blot analysis ofmarkers of the intrinsic mitochondrial apoptosis pathway. Increasingdoses of M2 led to PUMA induction (up to ˜17-fold increase in both celllines when treated with 40 μM M2) and progressive reduction of Bcl-2levels (undetectable in HCT-116 and ˜0.1-fold in HT-29 cells whentreated with 40 μM M2). A progressive increase in cytochrome c releasewith increasing doses of M2 (up to ˜2.2-fold increase in both cell lineswhen treated with 40 μM M2) was observed, as was a clear, progressivereduction in XIAP expression with increasing doses of M2 (undetectablein HCT-116 and ˜0.3-fold in HT-29 cells when treated with 40 μM M2).Finally, increasing concentration of the cleaved forms of caspase 9 (upto ˜4.6-fold increase in both cell lines when treated with 40 μM M2) andcaspase 3 (up to ˜3 to 4-fold increase in both cell lines when treatedwith 40 μM M2) were detected with increasing doses of M2. Without beingbound by theory, these results suggest that markers of the mitochondrialpathway (PUMA, Bcl-2) of apoptosis were activated upon exposure to M2,and ultimately led to the release of the corresponding apoptosis markers(cytochrome c, cleaved caspases 3 and 9).

8S, 10S, M2′ and M2″ Activate Apoptosis

Screening of the markers modulated by the pro-apoptotic activity of M2gave similar results for 8S and 10S and their respectivecysteine-conjugated metabolites M2′ and M2″. In other words, PUMA, andcleaved caspase-3 were all up-regulated, with concomitantdown-regulation of Bcl-2. The changes in markers expression was observedin both cell lines in a nearly identical amplitude between the parentcompound and its corresponding metabolite.

Shogaols and its Cysteine-Conjugated Metabolites Affect Wild-Type andMutant p53 Expression in Human Colon Cancer Cells HCT-116 and HT-29

To study the impact of M2 on p53 regulation in human colon cancer cellsand its dependency on p53 integrity, p53 wild-type HCT-116 or p53 mutantHT-29 cells were cultured with M2 or 6S for 24 hours at concentrationsof 10, 20, or 40 μM. The p53 response in colon cancer cells to 6Streatment (10, 20, or 40 μM for 24 hours) was observed in HCT-116 andHT-29. After M2 or 6S treatment, a dose-dependent up-regulation of p53was noted in both wild-type and mutant cancer cell lines, indicatingthat M2 or 6S regulation of p53 does not require a wild-type gene,although induction of p53 expression by M2 or 6S is dramatic in HCT-116cells and slightly less striking (but still significant) in HT-29 cells.The expression of p53 in HCT-116 and HT-29 cells after exposure to 20 μMof 8S, M2′, 10S or M2″ showed an increase in p53 accumulation for allcompounds. The cysteine-conjugated metabolites were able to increase p53accumulation in a similar manner to their parent compound.

6S and its Cysteine-Conjugated Metabolite M2 Affect Reactive OxygenSpecies Generation in Human Colon Cancer Cells HCT-116 and HT-29

As shown in FIGS. 15A and 15B, the trends of ROS induction by both M2and 6S are similar, with the greatest peak at 2 hours after treatmentand a steady decline thereafter, which is consistent with the changes ofglutathione levels in cancer cells. Treatment of HCT-116 cells with 40μM M2 produced the greatest ROS activity after two hours, with greaterthan 2-fold induction (with statistical significance, p<0.0001). Thescales of induction of ROS by M2 or 6S are parallel to the p53 inductionresponse in the two respective cell lines. That is, in HCT-116 cells,ROS activity is induced 2-fold or greater by M2 or 6S treatment, whilein HT-29 cells, ROS activity is induced less than 1.5-fold.

Treatment of HCT-116 and HT-29 cells for 24 hours with M2 or 6S (10, 20,or 40 μM) and supplemented with 5 mM GSH suppressed ROS accumulation inthe cells. Addition of GSH suppressed p53 induction in both cell linesfor all concentrations of M2 or 6S, suggesting that p53 does notaccumulate if there is no ROS generation.

M2 Inhibits Colony Formation in Human Colon Cancer Cells HCT-116 andHt-29

Human colon cancer cells HCT-116 (FIG. 16A) and HT-29 (FIG. 16B) weretreated with M2 with doses ranging from 0 to 40 μM for two weeks.Inhibition of colony formation was observed in a dose-dependent mannerin both cell lines, with 50% inhibition between 5 and 10 μM treatments.Following a trend underlined previously, HCT-116 cells are slightly moresensitive to M2 than HT-29 cells.

DISCUSSION

[6]-shogaol has been shown herein to be extensively metabolized in miceand in cancer cells. Reduction of xenobiotic carbonyls is a metabolicroute to produce more hydrophilic and often less toxic compounds, whichcan be substrates for phase II conjugation byUDP-glucuronosyltransferases or sulfotransferases, leading ultimately toexcretion of the products. As disclosed herein reduced metabolites wereformed in which M11 is the double-bond-reduced metabolite of[6]-shogaol, and M9 and M6 are ketone group-reduced metabolites of M11and [6]-shogaol, respectively.

The metabolic profiles of shogaols in mouse and in human urine wereanalyzed using liquid chromatography/electrospray ionization (ESI)tandem mass spectrometry. The structures of major metabolites (FIG. 1)were identified by analyzing the MS² and MS³ spectra of each compound.The regulation of GSH by [6]-shogaol was also investigated in humancolon cancer cells. In particular, the metabolism of [10]-shogaol inmouse urine, was investigated with special focus on the mercapturic acidpathway, and then the formation of thiol-conjugated metabolites ofshogaols ([6]-, [8]-, and [10]-shogaols) in human urine was studied.Eight major thiol-conjugated metabolites of [10]-shogaol were detectedin mouse urine, while six major thiol-conjugated metabolites of[6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and twothiol-conjugated metabolites of [10]-shogaol were detected in urinecollected from human after drinking ginger tea, using liquidchromatography/electrospray ionization tandem mass spectrometry. Withoutbeing bound by theory, the results indicate the mercapturic acid pathwayis a major metabolic route for [10]-shogaol in mice and for shogaols inhuman. The regulation of glutathione (GSH) by [6]-shogaol in HCT-116human colon cancer cells was also investigated; [6]-shogaol, afterinitially depleting glutathione levels, was shown to subsequentlyrestore and increase GSH levels over time.

M2 was shown to substantially retain the biological activities of[6]-shogaol, with an IC₅₀ of 24.43 μM in HCT-116 human colon cancercells and an IC₅₀ of 25.82 μM in H-1299 human lung cancer cells. M13 hadIC₅₀ values of 45.47 and 47.77 μM toward HCT-116 and H-1299 cells,respectively. The toxicity evaluation of the synthetic metabolites (M1,M2, and M4-M13) against human normal fibroblast colon cells CCD-18Co andhuman normal lung cells IMR-90 demonstrated a detoxifying metabolicbiotransformation of [6]-shogaol. The most active metabolite M2 hadalmost no toxicity to CCD-18Co and IMR-90 normal cells with IC₅₀ of99.18 and 98.30 μM, respectively. TUNEL (Terminal deoxynucleotidyltransferase dUTP nick end labeling) assay indicated apoptosis wastriggered by metabolites M2, M13, and its two diastereomers M13-1 andM13-2. There was no significant difference between the apoptotic effectof [6]-shogaol and those of M2 and M13 at 6 hour time point treatment.

Further, the in vitro metabolism of [6]-shogaol was compared among fivespecies using liver microsomes from mouse, rat, dog, monkey, and human.Following incubations with [6]-shogaol, three major reductivemetabolites 1-(4′-hydroxy-3′-methoxyphenyl)-4-decen-3-ol (M6),1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-ol (M9), and1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-one (M11), as well as two newoxidative metabolites(1E,4E)-1-(4′-hydroxy-3′-methoxyphenyl)-deca-1,4-dien-3-one (M14) and(E)-1-(4′-hydroxy-3′-methoxyphenyl)-dec-1-en-3-one (M15) were found inall species (See FIG. 1). The kinetic parameters of M6 in livermicrosomes from each respective species were quantified usingMichaelis-Menten theory. A broad CYP-450 inhibitor,1-aminobenzotriazole, precluded the formation of oxidative metabolitesM14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductaseinhibitor, eradicated the formation of the reductive metabolites M6, M9,and M11 in all species. Metabolites M14 and M15 were tested for cancercell growth inhibition and induction of apoptosis and both showedsubstantial activity, with M14 displaying greater potency than[6]-shogaol.

[6]-shogaol can be metabolized through the mercapturic acid pathway.Initial conjugation with GSH promoted by glutathione transferase givesrise to the corresponding conjugate, and the GSH conjugate undergoesfurther enzymatic modification: first modification by□-glutamyltranspeptidase to form the cysteinylglycine conjugate; thenalteration by cysteinyl-glycine dipeptidase or aminopeptidase M to formthe cysteine conjugate; and finally conversion by N-acetyltransferase toform the N-acetylcysteine conjugate. Both the cysteine and theN-acetylcysteine conjugates act as substrates of cysteine S-conjugate□-lyase, a mainly renal and hepatic enzyme that cleaves the S—C bond inthe cysteinyl moiety, thus liberating a thiolated metabolite, which canbe further S-methylated by thiol S-methyltransferase to form5-methylthio-1-(4″-hydroxy-3″-methoxyphenyl)-decan-3-one (M10) or5-methylthio-1-(4″-hydroxy-3″-methoxyphenyl)-decan-3-ol (M12).

Attention should be paid to the dose administrated as a supplement of acondensed ginger extract. The metabolism of [6]-shogaol in HCT-116 andHT-29 human colon cancer cells, H-1299 human lung cancer cells, andCL-13 mouse lung cancer cells has been evaluated. The results show that[6]-shogaol in cancer cells has a similar metabolic pathway as that inmice. 5-glutathionyl-[6]-shogaol in treated HCT-116 cells was detected,giving further evidence to the existence of the mercapturic acidpathway. However, secondary metabolites such as cysteinyl,N-acetylcysteinyl, and cysteinylglycinyl conjugates were not observed inthe cancer cell lines. Without being bound by theory, it is possibly dueto the absence of the enzymes that lead to the loss of the individualamino acids from the GSH conjugate of [6]-shogaol. Over time, thedouble-bond-reduced product (M11) formed and the ketone group of M11 wasreduced to form M9. At 24 h, [6]-shogaol was almost completely convertedto M9 and M11 in HCT-116 and H-1299 cells and to M9 in CL-13 cells.

M9 and M11 both exhibit measurable antiproliferative activity in HCT-116and H-1299 cancer cells, albeit with less potency than [6]-shogaol(FIGS. 2A and 2B). In addition, M9 and M11 are capable of triggeringapoptosis in human colon and lung cancer cells (FIGS. 2C and 2D).[6]-shogaol demonstrated a superior apoptotic effect, so M9 and M11 areat least partially implicated in the stimulation of apoptosis.

The growth inhibitory effects of the synthetic metabolites were comparedwith [6]-shogaol in two human cancer cells and two human normal cells.Two metabolites, M2 and M13, showed the most comparable growthinhibitory effects to [6]-shogaol towards cancer cells. M2 exhibited adiscriminatory effect, that is, it did not seem to be toxic towardsnormal cells. This effect was not detected with [6]-shogaol. M13 alsoshowed less toxic effects towards normal cells compared to [6]-shogaol.In addition, M5, M6 and M8-M12 also had certain potency against thegrowth of cancer cells, but showed no toxicity towards normal cells withIC₅₀ values greater than 100 □M (FIGS. 3 and 4). Metabolites of[6]-shogaol remain bioactive against cancer cells but are much lesstoxic than [6]-shogaol to normal cells.

As disclosed herein, TUNEL assay showed that both M2 and M13, but notM6, are capable to induce cancer cell apoptosis in both HCT-116 humancolon cancer cells and H-1299 human lung cancer cells (FIGS. 6A and 6B).For M13, apoptosis induction could not firmly be attributed to oneisomer or the other, suggesting that stereo configuration is notdeterminative to the bioactivity of this compound. Both M2 and M13 cantrigger apoposis in HCT-116 cells at a level similar to that of[6]-shogaol at the 6 hour time point (FIG. 6C). However, after 24 hoursof exposure to the metabolites, the percentage of TUNEL-positive cellswas mostly unchanged at 20 μM for both M2 and M13 while the effect of[6]-shogaol was increased. The induction effect of M2 on apoptosis at aconcentration of 40 μM increased at the 24 hour time point compared tothat of the 6 hour time point, which was higher than that of M13 at a 40μM concentration. A concentration-dependant effect of [6]-shogaol andits metabolites was observed on cancer cell apoptosis, where an increasein concentration of a compound resulted in a corresponding increasedpercentage of apoptotic cells.

The metabolite M13 is a mixture of M13-1 and M13-2 and had slightlybetter growth inhibitory effects on cancer cells than either of the twodiastereomers alone. The isomers had similar activity, though M13-2 wasslightly more potent than M13-1. M13 was identified as the metabolite of[6]-shogaol in the form of a mixture of M13-1 and M13-2 in HCT-116 humancolon cancer cells.

The metabolism of [6]-shogaol is comparable in liver microsomes frommouse, rat, dog, monkey, and human. The spectra were similar across allanimal species and the major metabolites were found in all samples, witha few differences—for instance, the ratios and relative abundances ofthe major metabolites were not conserved. Metabolite M6, which was thedominant peak in spectra from the rodent species after 30 minutesincubation of [6]-shogaol with microsomes, was found in an intermediaterelative amount in dog and in human, and was a minor metabolite inmonkey. Metabolite M11 was the major product of [6]-shogaol metabolismin HLM, but a very minor product of incubation from microsomes of otherspecies, with minute amounts in MLM and CyLM. The greatest speciesvariegation noted was metabolites M14 and M15: M14 was the majormetabolite in CyLM, while the peak produced from this compound was minorin all other species, with the smallest amount produced from HLMincubation; metabolite M15, although present in all species after[6]-shogaol metabolism, was very minor in mouse, rat, and human and wasabundant in dog and monkey.

Using a general CYP-450 inhibitor ABT, it was shown that both M14 andM15 were catalyzed predominantly by CYP-450 enzymes, suggesting that theP450 enzymes involved in [6]-shogaol metabolism are not conserved as afunction of evolutionary similarity, given the disparity between CyLMand HLM. Genetically, monkey and human have a low relative divergence;however, minute differences in the composition of the CYP enzymes inseemingly familiar species have apparent metabolic consequences.

An α,β-unsaturated ketone such as [6]-shogaol is likely to undergoselective reductive metabolism with initial chain saturation andsubsequent reduction to the alcohol metabolite by an aldo-ketoreductase. An easily accessible and well known dehydrogenase inhibitor,licorice root derivative 18β-glycyrrhetinic acid (18β-GA), was used toinvestigate the role of aldo-keto reductase enzymes in [6]-shogaolmetabolism in liver microsomes. Administration of 18β-GA inhibited[6]-shogaol metabolism in all species, thereby verifying the assumptionthat [6]-shogaol reductive metabolism was a function of aldo-ketoreductase(s).

To quantify a portion of [6]-shogaol metabolism, the pharmacokinetics ofmajor metabolite M6 formation in MLM, RLM, DLM, CyLM, and HLM wasinvestigated using Michaelis-Menten parameters. The calculated resultswere consistent with the observed spectra engendered from incubations ofeach respective species and [6]-shogoal. That is, mouse and rat showedsignificant preference for M6 formation, both qualitatively andquantitatively, with the largest peak areas for this metabolite and thelowest K_(m) values after 30 minute incubations with [6]-shogaol.Intrinsic clearance values for these species were also the highest,indicating greater catalytic efficiency. Conversely, monkey livermicrosomes produced a small amount of M6, with a matching high K_(m)value and a low intrinsic clearance value. Human liver microsomes gaveintermediate values for K_(m) and intrinsic clearance, compared to thefour species

The study of the metabolism of [6]-shogaol in different species providedthe opportunity to identify two oxidative metabolites,(1E,4E)-1-(4-Hydroxy-3-methoxyphenyl)deca-1,4-dien-3-one(M14) and(E)-1-(4-Hydroxy-3-methoxyphenyl)dec-1-en-3-one (M15) (FIG. 1).Bioactivity assays showed M14 had a significantly increased potency over[6]-shogaol, in both killing cancer cells and inducing apoptosis, whileM15 displayed moderate activity. Without being bound by theory, itappears that the increased potency of M14 can be attributed to theadditional double bond on the α,β-unsaturated ketone [6]-shogaol. Thedouble bonds in this compound are thought to be specifically availableto sulfhydryl groups via Michael addition and may react by depletingantioxidants such as glutathione. Drastic glutathione depletion istypically an indication of cellular distress and may induce apoptosis.Similarly, as M15 retains an α,β-unsaturated ketone composition similarto [6]-shogaol, it is likely the activity retained by this metabolitecan be attributed to this structure. However, in examining the placementof the double bond, the reduced activity is putatively a result ofsteric hindrance of the benzyl ring against bonding of sulfhydryl groupsand subsequent glutathione depletion. Metabolites M14 and M15 inducedapoptosis in these cancer cells lines (FIG. 8), indicative of theirmeans of effect against cancer cells.

The cysteine-conjugated 6S (M2), exhibits a cancer cell toxicity similarto the parent compound 6S, but is relatively less toxic towards normallung cells than 6S. Both compounds can cause cancer cell death by anactivation of the mitochondrial apoptotic pathway. The cancer celltoxicity is initiated by early modulation of glutathione (GSH)intracellular content; the generated oxidative stress activates a p53transcription-independent pathway that ultimately leads to the releaseof mitochondria-associated apoptotic molecules such as cytochrome C, andcleaved caspases 3 and 9. In a xenograft nude mouse model, a dose of 30mg/kg of 6S or M2 was able to significantly decrease tumor burden inanimals, without any associated toxicity to the animals. This effectcorrelated with an induction of apoptosis and reduction of cellproliferation in the tumors. 6S and M2 can activate a similar cascade ofpathways ultimately leading to cancer cell apoptosis, and that thecysteine-conjugated metabolite has a superior in-vivo cancerchemopreventive potential, in addition to its ability to discriminatebetween cancer and normal cells, while decreasing tumor burden.

While both compounds displayed a significant toxicity towards A549cancer cells in the MTT assay, M2 was less toxic towards normal cells,suggesting that the cysteine-conjugation of 6S allowed discriminationbetween cancerous and normal human lung cells.

6S and its metabolite M2 activate the apoptosis pathway in A549 cells,based on data of the detection of cytoplasmichistone-associated-DNA-fragments upon treatment with 6S or M2 for 24hours. While the IC₅₀ of M2 is higher than 6S, its capacity tospecifically induce apoptosis was superior. The final apoptotic markerssuch as cytochrome C, caspases 3 and 9 and their cleaved isoforms arealso modulated, while the markers of the Bcl-2 family (Bcl-2, Bak, Bax)were not affected. An early increase in p53 and PUMA, two major actorsin the transmission of cellular changes such as (but not limited to)oxidative stress, were observed. Treatment of A549 cells with 6S and M2led to a disturbance in GSH homeostasis, which would explain thegeneration of oxidative stress.

When the interaction between Bcl-2 or Bcl-XL and p53 is disrupted usingthe targeted chemical inhibitor pft, the toxicity of both 6S and M2 isreduced (FIGS. 12B and 12C), as was the induction of apoptosis (FIG.12D). Without being bound by theory, an apoptotic signal transmittedthrough the activation of the transcription-independent mitochondrialp53 pathway would explain the lack of variation in the Bcl-2 familymarkers expression. A marked increase in PUMA expression was observedafter 24 hours of exposure to both 6S and M2. PUMA is another moleculethat can mediate the p53 apoptotic message through its interaction withmembers of the Bcl-2 family, suggesting that apoptosis is in partaccountable for the death of A549 cells, and that multiple mechanismsinducing apoptosis and/or toxicity in cancer cell toxicity are actingtogether.

In the xenograft experiment, A549 xenograft cancer cell growth wassignificantly delayed by both 6S and M2. M2-treated animals showed afurther reduction in tumor burden compared to the animals treated with6S at an equivalent dose of 30 mg/kg body weight. As demonstrated byTUNEL staining of tumor tissues, 6S could still significantly induceapoptosis in-vivo, while M2 was close to significance. Both 6S and M2decrease cell proliferation. Only 6S at the higher dose was shown tohave a significant effect while M2 was close to significance. This isconsistent that blocking of the apoptotic pathway through chemicalmethods in-vitro only partially rescued cells from the toxicity of 6Sand M2. Similarly, the percentage of apoptotic cells detected by ELISAcould not be entirely restored to DMSO (control)-treated levels,especially at the higher concentration (40 μM) of the compounds, whentreated with pft.

Cysteine-conjugated shogaols (M2, M2′ and M2″) are the major metabolitesof [6]-, [8]-, and [10]-shogaol in humans. M2 is a carrier of its parentmolecule [6]-shogaol in cancer cells and is less toxic to normal colonfibroblast cells. [8]- and [10]-shogaol have similar metabolic profilesto [6]-shogaol and exhibit similar toxicity towards human colon cancercells. Analogously, M2′ and M2″ both show low toxicity against normalcolon cells but retain potency against colon cancer cells.Cysteine-conjugated shogaols cause cancer cell death through theactivation of the mitochondrial apoptotic pathway. Without being boundby theory, oxidative stress activates a p53 pathway that ultimatelyleads to PUMA induction, down-regulation of Bcl-2, followed bycytochrome c release, perturbation of inhibitory interactions of XIAPwith caspases, and finally caspase 9 and 3 activation and cleavage. Abrief screen of the markers attenuated by the proapoptotic activity ofM2 revealed similar results for [8]- and [10]-shogaols and theirrespective cysteine-conjugated metabolites M2′ and M2″.

An initial reaction between the α,β-unsaturated ketone functional groupof 6S and the cysteine sulfhydryl component of GSH takes place in theMAP, giving rise to the corresponding conjugates. The conjugates thenundergo series of enzymatic modifications on the GSH moiety, formingcysteinylglycine-, cysteine-, and finally N-acetylcysteine-conjugates.Both 8S and 10S are also metabolized in human through the MAP and thecysteine-conjugated metabolites, M2′ and M2″ respectively wereidentified as their major metabolites in human urine. These metabolismproducts are more water soluble and both less toxic and less pungentthan their parent compounds.

M2′ and M2″ were identified as the major metabolite of 8S and 10S,respectively, from humans upon consumption of ginger tea and are thecarriers of 8S and 10S, respectively. The metabolites have similaranti-proliferative activity against human colon cancer cells and lesstoxicity in normal human colon cells to their respective parentcompounds. This portion of Phase II metabolism transforms electrophilesto less reactive and more water-soluble intermediates, thus aiding intheir mobility and decreasing their toxicity en route.

M2 treatment has been shown to induce ROS generation that in turnup-regulated p53 expression and induced apoptosis through themitochondrial pathway. M2 induces apoptosis in both wild-type p53HCT-116 human colon cancer cells as well as mutant p53 HT-29 human coloncancer cells. Although the p53 pro-apoptotic pathway was exploited forat least some of M2's bioactivity, the metabolite's efficacy wasultimately not compromised by p53 mutation. Without being bound bytheory, this suggests that the cysteine-conjugated metabolite of 6Swould still be able to activate a p53 apoptotic response even in cancercells containing mutations of the p53 gene.

Both HCT-116 and HT-29 human colon cancer cells experienced a dramaticdown-regulation of Bcl-2 after M2 treatment. Interestingly, PUMA, atranscriptional target of p53, was also up-regulated in both coloncancer cell lines after treatment with M2. Upon atranscriptionally-induced signal from p53, PUMA assists in promotingapoptosis by disrupting the association restraints Bcl-xL exerts on p53,thus liberating the molecule to exert pro-apoptotic activity, butbinding to Bcl-xL in the process. This evidence supports the strong roleof M2 as a chemopreventive agent against colon cancer cells that inducesp53 expression and downstream regulation.

Treatment of colon cancer cells HCT-116 and HT-29 with M2 in this studylead to apoptosis, through early production of reactive oxygen species.Over-abundance of ROS combined with a cancer cell's reduceddetoxification ability often leads to oxidative stress sufficient toinduce programmed cell death. Apoptosis induced by p53 is at leastpartially dependent upon accumulation of ROS in the current model,suggesting that M2 causes p53 induction of apoptosis via ROS productionin both HCT-116 and HT-29 human colon cancer cells. Since it has beendemonstrated that metabolites M2′ and M2″ activate similar markers ofapoptosis and contain the same chemical reactivity as M2, it isreasonable to conclude that these metabolites behave in a similar wayand also activate cancer cell apoptosis through ROS induction and thesubsequent p53 accumulation. In addition, it was demonstrated that M2does not exclusively induce cancer cell death through apoptosis and canalso influence other major mechanisms such as cell proliferation; itfurther follows that other cysteine conjugated metabolites originatingfrom the same MAP have that potential as well. It has been shown thatcysteine-conjugated shogaols are novel compounds with a putative role asnatural pharmaceuticals with low-toxicity, high-potency, and at leastpartially indifferent to p53 integrity in colon cancer cells.

The patents and publications listed herein describe the general skill inthe art and are hereby incorporated by reference in their entireties forall purposes and to the same extent as if each was specifically andindividually indicated to be incorporated by reference. In the case ofany conflict between a cited reference and this specification, thespecification shall control. In describing embodiments of the presentapplication, specific terminology is employed for the sake of clarity.However, the invention is not intended to be limited to the specificterminology so selected. Nothing in this specification should beconsidered as limiting the scope of the present invention. All examplespresented are representative and non-limiting. The above-describedembodiments may be modified or varied, without departing from theinvention, as appreciated by those skilled in the art in light of theabove teachings. It is therefore to be understood that, within the scopeof the claims and their equivalents, the invention may be practicedotherwise than as specifically described.

What is claimed:
 1. A compound having the formula:

wherein n is selected from the group of 2, 4, 6 and combinationsthereof; and the compound is in an isolated or purified form.
 2. Thecompound of claim 1 as a pharmaceutically acceptable salt.
 3. Thecompound of claim 1 as a pharmaceutically acceptable hydrate.
 4. Apharmaceutical composition comprising a unit dose of an activeingredient and a pharmaceutical grade carrier wherein said activeingredient is a compound having the formula:

and wherein n is selected from the group consisting of 2, 4, 6 andcombinations thereof.
 5. The pharmaceutical composition of claim 4,wherein said active ingredient is a pharmaceutically acceptable salt orhydrate of said formula.
 6. The pharmaceutical composition of claim 4,wherein said compound is in an isolated or purified form.
 7. Anutraceutical composition comprising an active ingredient and a foodgrade carrier wherein said active ingredient is a compound having theformula:

and wherein n is selected from the group consisting of 2, 4, 6 andcombinations thereof.
 8. The nutraceutical composition of claim 7,wherein said active ingredient is a pharmaceutically acceptable salt orhydrate of said formula.
 9. The nutraceutical composition of claim 7,wherein said compound is in an isolated or purified form.
 10. Thenutraceutical composition of claim 7 wherein n consists essentially of 2and
 4. 11. The nutraceutical composition of claim 7 wherein n consistsessentially of 2 and
 6. 12. The nutraceutical composition of claim 7wherein n consists essentially of 4 and
 6. 13. The nutraceuticalcomposition of claim 7 wherein n consists essentially of 2, 4 and 6.